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PubMed1998

Interaction of troponin-H and glutathione S-transferase-2 in the indirect flight muscles of Drosophila melanogaster.

Drosophila indirect flight muscles (IFMs) contain a 35 kDa protein which cross-reacts with antibodies to the IFM specific protein troponin-H isoform 34 (TnH-34). Peptide fingerprinting and peptide sequencing showed that this 35 kDa protein is glutathione S-transferase-2 (GST-2). GST-2 is present in the asynchronous indirect flight muscles but not in the synchronous tergal depressor of the trochanter (jump muscle). Genetic dissection of the sarcomere showed that GST-2 is stably associated with the thin filaments but the presence of myosin is required to achieve the correct stoichiometry, suggesting that there is also an interaction with the thick filament. The two Drosophila TnHs (isoforms 33 and 34) are naturally occurring fusion proteins in which a proline-rich extension of approximately 250 amino acids replaces the 27 C-terminal residues of the muscle-specific tropomyosin II isoform. The proteolytic enzyme, Igase, cleaves the hydrophobic C-terminal sequence of TnH-34 at three sites and TnH-33 at one site. This results in the release of GST-2 from the myofibril. The amount of GST-2 stably bound to the myofibril is directly proportional to the total amount of undigested TnH. It is concluded that GST-2 in the thin filament is stabilized there by interaction with TnH. We speculate that the hydrophobic N-terminal region of GST-2 interacts with the hydrophobic C-terminal extension of TnH, and that both are close to a myosin cross-bridge.

Animals↗
PubMed1999

Sexual dimorphism in rat left atrial function and response to adrenergic stimulation.

A number of investigations in humans and animals suggest that there may be intrinsic sex-associated differences in cardiac function. Using left atrial preparations from male and female rat hearts, we examined differences in myocardial function and response to adrenergic agonists. Contractile parameters were measured in isolated atria by conventional isometric methods in the absence or presence of isoproterenol or phenylephrine. Responsiveness to Ca2+ was measured in detergent-skinned atrial fibers and actomyosin ATPase activity was measured in isolated myofibrils. Tetanic contractions were generated by treating the atrium with ryanodine followed by high frequency stimulation. Developed force was greater and maximal rates of contraction and relaxation were more rapid in the female atrium. The relationship between Ca2+ concentration and force in both intact atria and detergent-skinned atrial fibers in females fell to the left of that for males. At low Ca2+ concentrations, skinned fibers from female atria generated more force and myofibrils from female atria had higher myosin ATPase activity than males. Tetanic contraction in the presence of high extracellular Ca2+ was greater in female atria. Male atrium had larger inotropic responses to isoproterenol and to phenylephrine, but drug-elicited cAMP and inositol phosphate production did not differ between sexes. The results demonstrate sex-related differences in atrial function that can be partially explained by greater myofibrillar Ca2+-sensitivity in females. A potential contribution of sarcolemmal Ca2+ influx is suggested by greater tetanic contraction in ryanodine-treated female atrium. The larger response of males to adrenergic stimulation does not appear to be explained by higher production of relevant second messengers. Future studies will investigate the role of sex hormones in these sexually dimorphic responses and may indicate a need for gender-specific therapeutic interventions for myocardial dysfunction.

Adrenergic Agonists↗
PubMed2000

Cardiomyocyte cytoskeleton and myofibrillogenesis in healthy and diseased heart.

The unique cytoarchitecture of cardiomyocytes arises by complex interactions of different filamentous structures of the cytoskeleton. Intermediate filaments of the non-sarcomeric cytoskeleton are not essential for development but important for maintenance of myofibrils. Myofibrils consist of contractile proteins involved in force generation and the muscle cytoskeleton framework. The latter is essential for proper assembly and maintenance as well as for interaction with other cardiomyocytes or the extracellular matrix, thus being involved in force transmission. The information for sarcomere assembly is encoded in the proteins and some domains essential for faithful incorporation have been identified by epitope tagging experiments. Many KO mutations result in embryonic lethal phenotypes and new techniques e.g. using cardiomyocytes derived from ES cell-lines will have to be developed that allow to study such mutations in cardiomyocytes rather than whole organisms. Alterations in the expression levels of several proteins of the muscle cytoskeleton or impairment of their function by point mutations can result in increased mechanical stress in the cardiomyocytes which finally leads to cellular responses such as the development of dilated cardiomyopathy (DCM). MLP (muscle-LIM-protein) deficient mice develop DCM and changes in the mechanical coupling of cardiomyocytes result in alterations at the intercalated disks and enhanced accumulation of adherens junction proteins. Therefore, controlled interactions between proteins of the muscle cytoskeleton and contractile proteins are essential to ensure proper cardiac function and a more detailed insight in these processes might provide new tools to improve the contractile efficiency of the cardiomyocytes and thus working output in cardiomyopathies.

Animals↗
PubMed1981

Non-uniform ion distributions and electrical potentials in sarcoplasmic regions of skeletal muscle fibres.

We report here electrophysiological and ion distribution results obtained with mechanically skinned skeletal muscle fibres which indicate that a Donnan equilibrium, characterized by large ionic concentration and electrical potential differences, is established between the myofibrillar space and the rest of the sarcoplasm surrounding the myofibrils. The more negative electrical potential within the myofibrils (-15 to -29 mV for conditions similar to those in vivo) can strongly influence: (1) intracellular Ca2+ movements associated with contraction; (2) the location of sarcoplasmic enzymes, their substrates and products of reaction; (3) measurements of the electrical potential difference between the extra- and intracellular space and estimation of relative membrane permeabilities for certain ions.

Animals↗
PubMed2001

Actin dynamics at pointed ends regulates thin filament length in striated muscle.

Regulation of actin dynamics at filament ends determines the organization and turnover of actin cytoskeletal structures. In striated muscle, it is believed that tight capping of the fast-growing (barbed) ends by CapZ and of the slow-growing (pointed) ends by tropomodulin (Tmod) stabilizes the uniform lengths of actin (thin) filaments in myofibrils. Here we demonstrate for the first time that both CapZ and Tmod are dynamic on the basis of the rapid incorporation of microinjected rhodamine-labelled actin (rho-actin) at both barbed and pointed ends and from the photobleaching of green fluorescent protein (GFP)-labelled Tmod. Unexpectedly, the inhibition of actin dynamics at pointed ends by GFP-Tmod overexpression results in shorter thin filaments, whereas the inhibition of actin dynamics at barbed ends by cytochalasin D has no effect on length. These data demonstrate that the actin filaments in myofibrils are relatively dynamic despite the presence of capping proteins, and that regulated actin assembly at pointed ends determines the length of thin filaments.

Actin Cytoskeleton↗
PubMed2000

Association of plectin with Z-discs is a prerequisite for the formation of the intermyofibrillar desmin cytoskeleton.

Plectin is a high-molecular mass protein (approximately 500 kd) that binds actin, intermediate filaments, and microtubules. Mutations of the plectin gene cause a generalized blistering skin disorder and muscular dystrophy. In adult muscle, plectin is colocalized with desmin at structures forming the intermyofibrillar scaffold and beneath the plasma membrane. To study the involvement of plectin in myofibrillogenesis, we analyzed the spatial and temporal expression patterns of plectin in cultured differentiating human skeletal muscle cells and its relationship to desmin intermediate filaments during this process. Northern and Western blot analyses demonstrated that at least two different plectin isoforms are expressed at all developmental stages from proliferating myoblasts to mature myotubes. Using immunocytochemistry, we show that the localization of plectin dramatically changes from a network-like distribution into a cross-striated distribution during maturation of myocytes. Double immunofluorescence experiments revealed that desmin and plectin are colocalized in premyofibrillar stages and in mature myotubes. Interestingly, plectin was often found to localize to the periphery of Z-discs during the actual alignment of neighboring myofibrils, and an obvious cross-striated plectin staining pattern was observed before desmin was localized in the Z-disc region. We conclude that the association of plectin with Z-discs is an early event in the lateral alignment of myofibrils that precedes the formation of the intermyofibrillar desmin cytoskeleton.

Cells, Cultured↗
PubMed2006

Identification of Rho GTPases implicated in terminal differentiation of muscle cells in ascidia.

BACKGROUND INFORMATION: Members of the Rho GTPase family mediate changes in the actin cytoskeleton and are also implicated in developmental processes, including myogenesis. Nevertheless, a comprehensive analysis of these proteins during myofibrillogenesis has never been performed in any organism. RESULTS: Using the ascidian model to identify the role of Rho GTPases on myofibrillogenesis, we show that transcripts for all Rho GTPases are detected in muscle cells of the embryo. We find that activation of RhoA, TC10 and Cdc42 (cell division cycle 42) disturbs the polarity of muscle cells, whereas that of other Rho GTPases induced cell positioning defects. Moreover, dominant negative version of five Rho GTPases, RhoA, Rac2, RCL2 (Rac- and Cdc42-like 2), TC10 and WRCH (Wnt-1 responsive Cdc42 homologue), impaired the formation of mature myofibrils. CONCLUSIONS: Taken together, our results show that several Rho GTPase-dependent pathways are required to control the spatial localization of muscle cells in the embryo and to coordinate myofibril assembly. This stresses the importance of analysing the entire Rho family when studying a new biological process.

Animals↗
PubMed1981

C-protein from rabbit soleus (red) muscle.

A new form of skeletal-muscle C-protein has been isolated from rabbit soleus (red) muscle. This new form of C-protein has been purified to homogeneity by a procedure similar to that used to purify C-protein from white skeletal muscle. In soleus muscle, only this new form of C-protein could be detected, whereas in psoas (white) muscle, only the previously identified form of C-protein was detected. The content of C-protein in rabbit soleus muscle is comparable with that found in psoas muscle. Other rabbit skeletal muscles composed of a mixture of fibre types contained at least two forms of C-protein. C-Protein derived from red skeletal muscle bound to myosin isolated from either red or white tissue, with maximum binding occurring at a ratio of approximately 13 microgram of red C-protein/100 microgram of myosin. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate indicated that C-protein isolated from red skeletal muscle has a molecular weight approx. 7% greater than that of C-protein isolated from white skeletal muscle. The amino acid content of both forms of C-protein was similar but major differences in the mol % of isoleucine and threonine were found. Antiserum against C-protein from white rabbit skeletal muscle formed a single precipitin line with rabbit C-protein on double in agar. This antiserum did not form a precipitin line when diffused against red C-protein from rabbit skeletal muscle. Also, this antiserum bound specifically to the A-band region of myofibrils isolated from psoas (white) muscle, but it did not bind to myofibrils prepared from soleus (red) muscle.

Amino Acids↗
PubMed2000

Myocardial hypertrophy in transgenic mice overexpressing the bovine growth hormone (bGH) gene.

OBJECTIVES: The main purpose of the present study was to characterize cardiac muscle hypertrophy using both qualitative and quantitative microscopy in mice overexpressing the bovine growth hormone. RESULTS: Measurements of 30 fibres from each group revealed that fibre diameter in transgenic hearts was significantly larger than in control hearts. There was a significant decrease in interfibrillar space in transgenic hearts as compared with control hearts. The enlarged transgenic hearts displayed unchanged organelles such as normal myofibrils and mitochondria in a normal pattern, suggesting balanced growth. Myelin structures were occasionally observed between normal myofibrils. Moreover, myocardial beta-adrenergic receptors and muscarinic receptors in the hearts of transgenic mice overproducing GH were studied to see whether they are involved in the hypertrophic process. It was shown that the density of muscarinic receptors had decreased and the super-high affinity of muscarinic receptors was lost, without any significant changes in either the density or the affinity of beta-adrenergic receptors, as compared with controls. CONCLUSIONS: These results demonstrate that a GH excess was able to induce significant myocardial hypertrophy and that there was a downregulation of muscarinic receptors.

Animals↗
PubMed1997

Muscle patterning, differentiation and vascularisation in the chick wing bud.

Adult muscle is highly vascularised, with blood vessels being essential for adequate oxygenation of the tissue and for supporting increased metabolic demands. Whether this is the case during muscle development has not been examined. Resin histology was used to map the muscle splitting process and conventional transmission electron microscopy to examine early muscle differentiation at the midlimb level or later at the mid radius/ulna level in the chick wing bud from stages 24 (4.5 d) to 36 (10 d) (Hamburger & Hamilton, 1951). Microinjection of India Ink into the extra-embryonic vasculature was used to visualise the patent muscle microcirculation. The results showed that the premuscle masses are present at stage 24 and initial splitting of the muscle masses commences at stage 28. The final muscle pattern is not established until stage 36. At stage 26 the cells within the premuscle masses exhibited a mesenchymal morphology, but at stage 28 overt muscle differentiation was evident with myofibrils present within myoblasts. Undifferentiated mononucleated cells were interspersed with the differentiating myoblasts. The ratio of mononucleated cells:myoblasts decreased and the myoblasts became plumper and increasingly packed with myofibrils with age. There was no evidence of secondary myotube formation at any of the stages examined. Vascular invasion of the limb occurred at stage 35 just prior to the establishment of the final muscle pattern. This was surprising as it was assumed that myogenic differentiation would be both oxygen and nutrient dependent. The results of this study provide descriptions of the splitting of the premuscle masses through to the establishment of the final muscle pattern at the midlimb or mid radius/ulna level of the chick wing bud together with the differentiation of the myogenic cells within the developing muscles. However, the relationship between muscle patterning at the tissue level and muscle differentiation at the cellular level with vascularisation remains unclear. It is hoped that the results of the study may provide the basis for future investigations into mechanisms involved in muscle patterning and the signalling mechanisms for vascular invasion.

Animals↗
PubMed1988

[Congenital alcoholic cardiomyopathy in 3 children].

Three cases of congenital alcoholic cardiomyopathy and cardiac defects in children are described. The mothers were heavy spirits drinkers. One child had a ventricular septal defect, two had Fallot's tetralogy, all requiring operation. At open-heart surgery left-ventricular myocardial biopsies were obtained in all three children. Histological and electronmicroscopic examination revealed primary toxic and hypotrophic changes, which differed from those seen in alcoholic cardiomyopathy of the adult. The cells, their nuclei and the myofibrils had reduced diameters. The mitochondria were damaged to differing extent. The myofibrils were arranged in parallel and some had contraction bands and ruptures. The sarcoplasmic reticulum was dilated and had vacuoles. The cell surface penetrated into the interstitial tissue. All these changes could have resulted from inhibited embryofetal cell growth and cytotoxic damage by alcohol during pregnancy. It would seem that alcoholic cardiomyopathy occurs only in extreme forms of alcohol abuse during pregnancy. The prognosis is as yet unknown.

Cardiomyopathy, Alcoholic↗
PubMed1995

Ultrastructural and functional features of the developing mammalian heart: a brief overview.

The heart undergoes marked ultrastructural alterations during fetal and postnatal development. Early in fetal development, cardiac myocytes contain abundant pools of glycogen, scattered mitochondria and sparse, peripheral myofibrils. Transverse tubules are absent, and sarcoplasmic reticulum and intercalated discs are poorly developed. During late fetal and early postnatal development, myofibrils extend into the myocyte interior and attain a mature appearance, and the glycogen pools are reduced in size. In addition, transverse tubules develop and the morphological appearance of the sarcoplasmic reticulum and intercalated disc becomes increasingly complex. Experimental studies in sheep, corroborated by clinical studies in humans, also point to marked functional changes during development. In the fetus, the right ventricle is the dominant pumping chamber because right ventricular output exceeds left ventricular output, while pulmonary arterial and aortic pressures are similar. This functional difference is reflected in myocardial blood flow patterns, with blood flow to the right ventricle exceeding that to the left ventricle. The ventricular outputs equalize after birth, but a functional left ventricular dominance rapidly emerges following a postnatal increase in systemic vascular resistance and a decrease in pulmonary vascular resistance. This postnatal switchover in functional dominance is accompanied by a corresponding alteration in the relative level of ventricular myocardial blood flows. Consistent with right ventricular dominance in utero, myocytes in the right ventricle of the fetal sheep are larger and contain more myofibrillar material than those in the left ventricle. Left ventricular myocytes become larger than right ventricular myocytes after birth, but this adaptation to altered postnatal haemodynamics requires some weeks to become fully established.

Animals↗
PubMed1981

Myosin light chains and the developmental origin of fast muscle.

Physiological characteristics of embryonic and fetal fast muscle function are similar to those of adult slow muscles, whereas most biochemical data suggest that embryonic and fetal fast muscles contain only fast muscle myosin. In the studies reported here, myofibrillar preparations from developing avian pectoral muscle (fast muscle) were isolated and analyzed for myosin light-chain type and synthesis. These analyses show that early in development avian fast muscle synthesizes and assembles myofibrils with light chains of both slow and fast myosins. Later in development, fast muscle no longer assembles myofibrils containing slow myosin light chains due to the cessation of synthesis of slow myosin light chains in mid-development. These in vivo studies indicate that the more developmentally primitive type of skeletal muscle is one that synthesizes both slow and fast myosin light chains independent of its anatomic location, and an event(s) late in fast muscle development results in the repression of synthesis of slow myosin light chains.

Aging↗
PubMed1988

Three-dimensional immunogold localization of alpha-actinin within the cytoskeletal networks of cultured cardiac muscle and nonmuscle cells.

The ultrastructural distribution of alpha-actinin was studied in cultured hamster heart cells by immunogold replica electron microscopy. This technique enabled us to localize alpha-actinin within the cytoskeletal networks at high resolution and in three dimensions. Colloidal gold, indicating the presence of alpha-actinin, was localized on the Z bands of nascent myofibrils in myocytes and on stress fiber bundles in nonmuscle cells. alpha-Actinin staining was also seen on stellate foci, where cytoskeletal filaments converged along the inner myocyte cell membranes. Intermediate filaments were associated with Z bands of myofibrils, stress fibers, and subplasmalemmal actin networks at the specific points where alpha-actinin was localized on these structures. Heavy meromyosin treatment prior to immunostaining confirmed that the thin filaments contained actin. These results suggest that alpha-actinin serves to interlink these various cytoskeletal elements. In addition, this protein may be involved in the initial phases of filament organization during myofibrillogenesis along the inner surface of the myocyte plasma membrane.

Actinin↗
PubMed1990

Disruption of microfilament organization in living nonmuscle cells by microinjection of plasma vitamin D-binding protein or DNase I.

Plasma vitamin D-binding protein (DBP), which binds to monomeric actin, causes the breakdown of stress fibers when it is microinjected into nonmuscle cells. Disruption of the stress fiber network is also accompanied by shape changes in the cell that resemble those seen after cytochalasin treatment. When DBP was coinjected with fluorescently labeled alpha-actinin, no fluorescent stress fibers or attachment plaques were visible 30 min after injection. Twelve hours later the cells regained their flattened shape and their stress fibers. Fluorescently labeled DBP causes the same reversible changes in cell shape as the unlabeled protein. Upon injection, the labeled DBP diffuses throughout the cytoplasm, becoming localized by 12 hr in a punctate pattern, presumably due to lysozomal sequestration. Similar injections of DBP into skeletal myotubes and cardiac myocytes did not lead to shape changes or breakdown of nascent and/or fully formed myofibrils, even though DBP has a 2-fold higher binding affinity for muscle actin over that of the nonmuscle isoactins. Similar differential effects in nonmuscle cells were also observed after the microinjection of DNase I, another protein capable of binding monomer actin. The effects of these microinjected monomer actin-binding proteins imply that an accessible pool of monomer actin is needed to maintain stress fiber integrity in nonmuscle cells but not the integrity of the nascent or fully formed myofibrils in muscle cells.

Actin Cytoskeleton↗
PubMed1992

Association of titin and myosin heavy chain in developing skeletal muscle.

To understand molecular interactions that organize developing myofibrils, we examined the biosynthesis and interaction of titin and myosin heavy chain in cultures of developing muscle. Use of pulse-labeling, immunoprecipitation, and a reversible cross-linking procedure demonstrates that within minutes of synthesis, titin and myosin heavy chain can be chemically cross-linked into very large, detergent-resistant complexes retaining many features of intact myotubes. These complexes, predominantly of titin and myosin, occur very early in myofibrillogenesis as well as later. These data suggest that synthesis and assembly of titin and myosin are temporally and spatially coordinated in nascent myofibrils and support the hypothesis that titin molecules help to organize sarcomere formation.

Animals↗
PubMed1996

Importance of the ATP-ubiquitin-proteasome pathway in the degradation of soluble and myofibrillar proteins in rabbit muscle extracts.

Recent studies have suggested that activation of the ubiquitin-proteasome pathway is primarily responsible for the rapid loss of muscle proteins in various types of atrophy. The present studies were undertaken to test if different classes of muscle proteins are degraded by this pathway. In extracts of rabbit psoas muscle, the complete degradation of soluble proteins to amino acids was stimulated up to 6-fold by ATP. Peptide aldehyde inhibitors of the proteasome or the removal of proteasomes markedly inhibited only the ATP-dependent process. Addition of purified myosin, actin, troponin, or tropomyosin to these extracts showed that these proteins served as substrates for the ubiquitin-proteasome pathway. By contrast, degradation of myoglobin did not require ATP, proteasomes, or any known proteases in muscles. When myosin, actin, and troponin were added as actomyosin complexes or as intact myofibrils to these extracts, they were not hydrolyzed at a significant rate, probably because in these multicomponent complexes, these proteins are protected from degradation. Accordingly, actin (but not albumin or troponin) inhibited the degradation of 125I-myosin, and actin was found to selectively inhibit ubiquitin conjugation to 125I-myosin. Also, the presence of tropomyosin inhibited the degradation of 125I-troponin. However, neither actin nor tropomyosin inhibited the degradation of 125I-lysozyme or soluble muscle proteins. Thus, specific interactions between the myofibrillar proteins appear to protect them from ubiquitin-dependent degradation, and the rate-limiting step in their degradation is probably their dissociation from the myofibril.

Adenosine Triphosphate↗
PubMed1997

Functional equivalence of creatine kinase isoforms in mouse skeletal muscle.

Creatine kinase (CK) is a highly conserved enzyme abundant in skeletal muscle that has a key role in high energy phosphate metabolism. The localization of the muscle isoenzyme of CK (MM-CK) to the M line and the sarcoplasmic reticulum of myofibrils has been suggested to be important for proper force development in skeletal muscle. The importance of this subcellular compartmentation has not been directly tested in vivo. To test the role of myofibrilar localization of CK, the consequences of a complete CK isoform switch from MM-CK to the brain (BB-CK) isoform, which does not localize to the M line, was studied in transgenic mouse skeletal muscle. In MM-CK knockout mice there are large contractile defects. When MM-CK was replaced by BB-CK, the aberrant contractile phenotypes seen in MM-CK knockout mice were returned to normal despite the lack of myofibrillar localization. These results indicate that CK compartmentation to the myofibril of skeletal muscle is not essential for contractile function and that there is functional equivalence of creatine kinase isoforms in supporting cellular energy metabolism.

Animals↗