Role of calpains in promoting desmin filaments depolymerization and muscle atrophy

Muscle atrophy is an inevitable sequel of fasting, denervation, aging, exposure to microgravity, and many human diseases including, cancer, type-2 diabetes, and renal failure. During atrophy the destruction of the muscle's fundamental contractile machinery, the myofibrils, is accelerated leading to a reduction in muscle mass, weakness, frailty, and physical disability. Recent findings indicate that atrophy can be a major cause of death in affected individuals, and inhibition of muscle wasting is likely to prolong survival. Major advances in our understanding of the mechanisms for myofibril breakdown in atrophy include the discovery of biological pathways and key components that play prominent roles. On fasting or denervation, degradation of myofibrillar proteins requires an initial dissociation of the desmin cytoskeleton, whose integrity is critical for myofibril stability. This loss of desmin filaments involves phosphorylation, ubiquitination, and subsequent depolymerization by calpain-1, and appears to reduce myofibrils integrity and facilitate their destruction. Consequently, depolymerization of desmin filament in atrophy seems to be an early key event for overall proteolysis. A focus of this review is to discuss these new insights and the specific role of calpain-1 in promoting desmin filaments loss, and to highlight important key questions that merit further study.     

Bundle formation of smooth muscle desmin intermediate filaments by calponin and its binding site on the desmin molecule

Smooth muscle basic calponin, a major actin-, tropomyosin-, and calmodulin-binding protein, has been examined for its ability to interact with desmin intermediate filaments from smooth muscle cells using sedimentation analysis, turbidity changes, chemical cross-linking, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF/MS), and electron microscopic observations. Calponin interacted with desmin intermediate filaments in a concentration-dependent manner in vitro. The binding of calponin to desmin produced dense aggregates at 30 degrees C. The dense aggregates were observed by electron microscopy to be composed of large anisotropic bundles of desmin filaments, indicating that calponin forms bundles of desmin filaments. The addition of calmodulin or S100 to the mixture of calponin and desmin caused the removal of calponin from the desmin filaments and inhibited bundle formation in the presence of Ca(2+), but not in the presence of EGTA. Calponin-related proteins including G-actin, tropomyosin, and SM22, had little effect on the binding of calponin to desmin filaments, whereas tubulin weakly inhibited the binding. Desmin had little influence on the calponin-actin and calponin-tubulin interactions using the zero-length cross-linker, EDC. Domain mapping with chymotryptic digestion showed that the binding site of calponin resides within the central a-helical rod domain of the desmin molecule. The chemical cross-linked products of calponin and synthetic peptides (TQ27, TNEKVELQELNDRFANYIEKVRFLEQQ; EE24, EEELRELRRQVDALTGQRARVEVE) derived from the rod domain were detected by MALDI TOF/MS. Furthermore, the calponin-desmin interaction was significantly inhibited by the addition of EE24, but only slightly by TQ27. These results suggest that calponin may act as a cross-linking protein between desmin filaments as well as among intermediate filaments, microfilaments and microtubules in smooth muscle cells.     

Arrangement of desmin intermediate filaments in smooth muscle cells as shown by high-resolution immunocytochemistry

To gain additional information about the arrangement of intermediate filaments (IF) in normal smooth muscle, fresh avian gizzard was processed for immunoelectron microscopy. The protein A-gold immunocytochemical technique was applied for the localization of desmin antigenic sites. Desmin-containing IFs were located in an axial bundle that partially surrounds the nucleus and were associated with numerous mitochondria near the poles of the nucleus. The bundle probably extends the length of the cell. Antibody labeling also showed concentrations of IF around and between cytoplasmic dense bodies (CDB) and also between CDB and membrane-associated dense bodies (MADB). The relationship between the axial bundle and the nucleus and associated mitochondria suggests that the bundle may support and define the position of these organelles in the cell. A fraying or branching of the bundle may integrate the bundle into the remaining cytoskeletal network of the cell.     

Elastic filaments in skeletal muscle revealed by selective removal of thin filaments with plasma gelsolin

Muscle needs an elastic framework to maintain its mechanical stability. Removal of thin filaments in rabbit skeletal muscle with plasma gelsolin has revealed the essential features of elastic filaments. The selective removal of thin filaments was confirmed by staining with phalloidin-rhodamine for fluorescence microscopy, examination of arrowhead formation with myosin subfragment 1 by electron microscopy, and analysis by SDS-PAGE. Thin section electron microscopy revealed the elastic fine filaments (approximately 4 nm in diameter) connecting thick filaments and the Z line. After removal of thin filaments, both rigor stiffness and active tension generation were lost, but the resting tension remained. These observations indicate that the thin filament-free fibers maintain a framework composed of the serial connections of thick filaments, the elastic filaments, and the Z line, which gives passive elasticity to the contractile system of skeletal muscle. The resting tension that remained in the thin filament-free fibers was decreased by mild trypsin treatment. The only protein component that was digested in parallel with the decrease in the resting tension and the disappearance of the elastic filaments was alpha-connectin (also called titin 1), which was transformed from the alpha to the beta form (from titin 1 to 2, respectively). Thus, we conclude that the main protein component of the elastic filaments is alpha-connectin (titin 1).     

Distribution of vimentin and desmin filaments in smooth muscle tissue of mammalian and avian aorta

The presence of intermediate filament proteins in vascular tissue cells has been examined by immunofluorescence microscopy on frozen sections of the aortic wall of diverse vertebrates (rat, cow, human and chicken) and by gel electrophoresis of cytoskeletal proteins from whole aortic tissue or from stripped tunica media of cow and man. Most cells of the aortic wall in these species contain vimentin filaments, including smoooth muscle cells of the tunica media. In addition, we have observed aortic cells that are positively stained by antibodies to desmin. The presence of desmin in aortic tissue has also been demonstrated by gel electrophoresis for rat, cow and chicken. In aortic tissue some smooth muscle cells contain both types of intermediate filament proteins, vimentin and desmin. Bovine aorta contains, besides cells in which vimentin and desmin seem to co-exist, distinct bundles of smooth muscle cells, located in outer regions of the tunica media, which contain only desmin. The results suggest that (i) intermediate-sized filaments of both kinds, desmin and vimentin, can occur in vascular smooth muscle in situ and (ii) smooth muscle cells of the vascular system are heterogeneous and can be distinguished by their intermediate filament proteins. The finding of different vascular smooth muscle cells is discussed in relation to development and differentiation of the vascular system.     

Activation of rabbit muscle phosphofructokinase by F-actin and reconstituted thin filaments

Striking effects of F-actin and the reconstituted thin filament of muscle on the catalytic activity of rabbit muscle phosphofructokinase are demonstrated through direct measurements of enzymatic activity by using the pH stat. The addition of F-actin to solutions of phosphofructokinase at low ionic strength (10 mM KCl and 5 mM MgCl2) partially reverses the inhibition of the enzyme seen at high ATP concentrations and increases the apparent affinity of the enzyme for fructose 6-phosphate with slight effect on Vmax. F-Actin augments the activation of the enzyme obtained with AMP and partially counters the inhibition obtained with citrate. The maximum effect in the reversal of ATP inhibition is about the same for combinations of either F-actin or the thin filament with AMP as it is for AMP alone. In general, the effect of F-actin on the catalytic activity of phosphofructokinase is larger than that of the thin filament. The activation of phosphofructokinase by F-actin persists at physiological ionic strength.     

Visualization of dynamic simulations of muscle thin filaments

Following a novel computational formalism, the thin filament of muscle can be modeled by a computational machine containing a large number of finite automata that have one-to-one correspondence with the constituent protein molecules.¹ Computer graphics can be used to visualize the correspondence between the states of finite automata and the configurations of protein molecules according to the structural data. The dynamic simulation of the muscle filament that corresponds to the concurrent state transitions of finite automata can be represented as a sequence of video images. The kinetic and structural knowledge of individual protein molecules is, therefore, integrated into a coherent and functional system. This type of computation and visualization can also be useful for the investigation of molecular structure, function, and interaction in various complex biological systems.     

Role of desmin filaments in chicken cardiac myofibrillogenesis

Desmin filaments are muscle-specific intermediate filaments located at the periphery of the Z-discs, and they have been postulated to play a critical role in the lateral registration of myofibrils. Previous studies suggest that intermediate filaments may be involved in titin assembly during the early stages of myofibrillogenesis. In order to investigate the putative function of desmin filaments in myofibrillogenesis, rabbit anti-desmin antibodies were introduced into cultured cardiomyocytes by electroporation to perturb the normal function of desmin filaments. Changes in the assembly of several sarcomeric proteins were examined by immunofluorescence. In cardiomyocytes incorporated with normal rabbit serum, staining for alpha-actinin and muscle actin displayed the typical Z-line and I-band patterns, respectively, while staining for titin with monoclonal anti-titin A12 antibody, which labels a titin epitope at the A-I junction, showed the periodic doublet staining pattern. Staining for C-protein gave an amorphous pattern in early cultures and identified A-band doublets in older cultures. In contrast, in cardiomyocytes incorporated with anti-desmin antibodies, alpha-actinin was found in disoriented Z-discs and the myofibrils became fragmented, forming mini-sarcomeres. In addition, titin was not organized into the typical A-band doublet, but appeared to be aggregated. Muscle actin staining was especially weak and appeared in tiny clusters. Moreover, in all ages of cardiomyocytes tested, C-protein remained in the disassembled form. The present data suggest the essential role of desmin in myofibril assembly.     

Copurification of actin and desmin from chicken smooth muscle and their copolymerization in vitro to intermediate filaments

Desmin is a 50,000-mol wt protein that is enriched along with 100-A filaments in chicken gizzard that has been extracted with 1 M KI. Although 1 M KI removes most of the actin from gizzard, a small fraction of this protein remains persistently insoluble, along with desmin. The solubility properties of this actin are the same as for desmin: they are both insoluble in high salt concentrations, but are solubilized at low pH or by agents that dissociate hydrophobic bonds. Desmin may be purified by repeated cycles of solubilization by 1 M acetic acid and subsequent precipitation by neutralization to pH 4. During this process, a constant nonstoichiometric ratio of actin to desmin is attained. Gel filtration on Ultrogel AcA34 in the presence of 0.5% Sarkosyl NL-97 reveals nonmonomeric fractions of actin and desmin that comigrate through the column. Gel filtration on Bio-Gel P300 in the presence of 1 M acetic acid reveals that the majority of desmin is monomeric under these conditions. A small fraction of desmin and all of the actin elute with the excluded volume. When the acetic acid is removed from actin-desmin solutions by dialysis, a gel forms that is composed of filaments with diameters of 120-140 A. These filaments react uniformly with both anti-actin and anti-desmin antiserum. These results suggest that desmin is the major subunit of the muscle 100-A filaments and that it may form nonstoichiometric complexes with actin.     

Structural changes in thin filaments of crab striated muscle.

Low angle X-ray diffraction patterns were recorded from crab leg muscle in living resting state and in rigor (glycerol-extracted). Both resting and rigor patterns showed a series of layer-lines arising from a helical arrangement of actin subunits in the thin filaments. In the resting state, the crossover repeat of the long-pitch actin helices was 36.6 nm, and the symmetry of the genetic actin helix was an intermediate between $\text{26}\text{12}$ and $\text{28}\text{13}$. When the muscle went into rigor, the crossover repeat changed to 38.3 nm and the helical symmetry to $\text{28}\text{13}$.In the living resting pattern, six other reflections were observed on the meridian and in the near-meridional region. These were indexed as orders of 2 × 38.2 nm and could be assigned to troponin molecules; the spacings and the intensity distributions of these reflections could be explained by the model proposed by Ohtsuki (1974) for the arrangement of troponin molecules in the thin filaments.The muscle in rigor gave meridional and near-meridional reflections at orders of 2 × 38.3 nm. These were identified as the same series of reflections as was assigned to troponin in the living resting pattern, but were more intense and could be seen up to higher orders. We consider that the myosin heads attached to the thin filament at regular intervals along its axis also contribute to these reflections in the rigor pattern.     

Single particle analysis of relaxed and activated muscle thin filaments

The movement of tropomyosin from actin's outer to its inner domain plays a key role in sterically regulating muscle contraction. This movement, from a low Ca2+ to a Ca2+-induced position has been directly demonstrated by electron microscopy and helical reconstruction. Solution studies, however, suggest that tropomyosin oscillates dynamically between these positions at all Ca2+ levels, and that it is the position of this equilibrium that is controlled by Ca2+. Helical reconstruction reveals only the average position of tropomyosin on the filament, and not information on the local dynamics of tropomyosin in any one Ca2+ state. We have therefore used single particle analysis to analyze short filament segments to reveal local variations in tropomyosin behavior. Segments of Ca2+-free and Ca2+ treated thin filaments were sorted by cross-correlation to low and high Ca2+ models of the thin filament. Most segments from each data set produced reconstructions matching those previously obtained by helical reconstruction, showing low and high Ca2+ tropomyosin positions for low and high Ca2+ filaments. However, approximately 20% of segments from Ca2+-free filaments fitted best to the high Ca2+ model, yielding a corresponding high Ca2+ reconstruction. Conversely, approximately 20% of segments from Ca2+-treated filaments fitted best to the low Ca2+ model and produced a low Ca2+ reconstruction. Hence, tropomyosin position on actin is not fixed in either Ca2+ state. These findings provide direct structural evidence for the equilibration of tropomyosin position in both high and low Ca2+ states, and for the concept that Ca2+ controls the position of this equilibrium. This flexibility in the localization of tropomyosin may provide a means of sterically regulating contraction at low energy cost.     

Diagnosis of human childhood rhabdomyosarcoma of antibodies to desmin, the structural protein of muscle specific intermediate filaments

Four embryonal rhabdomyosarcomas, one tumor diagnosed as an undifferentiated sarcoma, probably a rhabdomyosarcoma, and six different non-muscular sarcomas were investigated with antibodies specific for different intermediate filament types. The tumor cells in the rhabdomyosarcomas and the undifferentiated tumor were stained clearly by antibodies to desmin, the intermediate filament type characteristic of muscle. The staining of tumor cell by antibodies to vimentin, the intermediate filament type characteristic of certain cell types of mesenchymal origin including myoblasts, was different in these 5 cases. In one case of embryonal rhabdomyosarcoma nearly all tumor cells were stained, but in the remaining cases few or no tumor cells were positive with the vimentin antibody. In these rhabdomyosarcomas not only the large rhabdomyoblasts, but also the small undifferentiated cells were labeled by antibodies to desmin. In the latter cell type the desmin filaments were arranged typically in coils. In contrast, tumor cells in the non-muscular mesenchymal sarcomas were stained only by antibodies to vimentin but not by antibodies to desmin or prekeratin. The retention of the desmin marker characteristic of normal muscle in cases of rhabdomyosarcoma not only allowed the undifferentiated desmin-positive sarcoma to be classified as rhabdomyosarcoma but also suggests that the use of antibodies to desmin could be very helpful in the future for the diagnosis of undifferentiated rhabdomyosarcomas.     

Evidence that nebulin is a protein-ruler in muscle thin filaments

Partial amino acid sequence was obtained from the massive myofibrillar protein nebulin. This consists of repeating motifs of about 35 residues and super-repeats of 7 x 35 = 245 residues. The repeat-motifs are likely to be largely alpha-helical and to interact with both actin and tropomyosin in thin filaments. Nebulin from different species was found to vary in size in proportion to filament length. The data are consistent with the proposal that nebulin acts as a protein-ruler to regulate precise thin filament assembly.     

Skeletal muscle atrophy leads to loss and dysfunction of muscle precursor cells

Atrophy of skeletal muscle leads to decreases in myofiber size and nuclear number; however, the effects of atrophic conditions on muscle precursor cells (MPC) are largely unknown. MPC lie outside myofibers and represent the main source of additional myonuclei necessary for muscle growth and repair. In the present study, we examined the properties of MPC after hindlimb suspension (HS)-induced atrophy and subsequent recovery of the mouse hindlimb muscles. We demonstrated that the number of MPC in atrophied muscles was decreased. RT-PCR analysis of cells isolated from atrophied muscles indicated that several mRNA characteristic of the myogenic program in MPC were absent. Cells isolated from atrophied muscles failed to properly proliferate and undergo differentiation into multinucleated myotubes. Thus atrophy led to a decrease in MPC and caused dysfunction in those MPC that remained. Upon regrowth of the atrophied muscles, these deleterious effects were reversed. Our data suggest that preventing loss or dysfunction of MPC may be a new pharmacological target during muscle atrophy. satellite cells; hindlimb suspension; proliferation; differentiation; myotubes     

Partial activation of thin filaments in resting frog skeletal muscle fibers

X-ray patterns from frog skeletal muscles at rest show a series of relatively weak meridional reflections which may be indexed as the 5, 7, 9, 11 and 13 orders of the repeat period of about 2 x 385 A. According to the model of the thin filaments structure, suggested by V. V. Lednev and G. M. Frank (1977), this period is specific for the activated or "switched on" state of the actin--containing filaments. At the same time, according to the generally accepted model (suggested in 1972), the axial repeat period of the thin filament structure is approximately equal to 385 A and does not depend on the functional state of the muscle. The existence of the repeat period of about 2 x 385 A in the thin filaments of a resting muscle suggests that even at rest the thin filaments of vertebrate skeletal muscle are not completely inhibited. It may be suggested that partial activation of the thin filaments in a resting muscle is the result of formation of long life rigorlike crossbridges, the existence of which was postulated by D. K. Hill in 1968 on the basis of his studies on resting tension in the frog skeletal muscle.     

Reconstitution of the muscle thin filament from recombinant troponin components and the native thin filaments

We have developed a technique by which muscle thin filaments are reconstituted from the recombinant troponin components and the native thin filaments. By this technique, the reconstituted troponin complex is exchanged into the native thin filaments in the presence of 20% glycerol and 0.3 M KCl at pH 6.2. More than 90% of endogenous troponin complex was replaced with the recombinant troponin complex. Structural integrity and Ca²⁺ sensitivity of the reconstituted thin filament prepared by this technique was confirmed by X-ray fiber diffraction measurements and the thin filament-activated myosin subfragment 1 ATPase measurements, respectively.     

Clinical and genetic heterogeneity in nemaline myopathy--a disease of skeletal muscle thin filaments

The term nemaline myopathy (NM) encompasses a heterogeneous group of disorders of primary skeletal muscle weakness characterized by the presence of nemaline rods in muscles of affected individuals. Disease severity is variable and unpredictable, with prognosis ranging from neonatal death to almost normal motor function. Recent advances in the identification of NM disease genes demonstrate that NM is a disease of the skeletal muscle sarcomere and, in particular, of the thin filaments. These findings are starting to alter the approach that neurologists and geneticists take to diagnosing and counseling patients with NM, and could lead to insights into specific directed therapies in the future.