Desmin Mutation in the C-Terminal Domain Impairs Traction Force Generation in Myoblasts

The cytoskeleton plays a key role in the ability of cells to both resist mechanical stress and generate force, but the precise involvement of intermediate filaments in these processes remains unclear. We focus here on desmin, a type III intermediate filament, which is specifically expressed in muscle cells and serves as a skeletal muscle differentiation marker. By using several complementary experimental techniques, we have investigated the impact of overexpressing desmin and expressing a mutant desmin on the passive and active mechanical properties of C2C12 myoblasts. We first show that the overexpression of wild-type-desmin increases the overall rigidity of the cells, whereas the expression of a mutated E413K desmin does not. This mutation in the desmin gene is one of those leading to desminopathies, a subgroup of myopathies associated with progressive muscular weakness that are characterized by the presence of desmin aggregates and a disorganization of sarcomeres. We show that the expression of this mutant desmin in C2C12 myoblasts induces desmin network disorganization, desmin aggregate formation, and a small decrease in the number and total length of stress fibers. We finally demonstrate that expression of the E413K mutant desmin also alters the traction forces generation of single myoblasts lacking organized sarcomeres.     

Coexpression of α-sarcomeric actin, α-smooth muscle actin and desmin during myogenesis in rat and mouse embryos I. Skeletal muscle

Expression of vimentin, desmin, alpha-sarcomeric and alpha-smooth muscle actins in embryonic tissues of rat and mice was examined using an immunohistochemical approach. The results showed a similarity in the expression of desmin and alpha-actin isoforms (alpha-sr and alpha-sm) in skeletal muscle cells during murine feto-embryonic development. In the two species, coexpression of alpha-sr and alpha-sm actins has been observed in cardiomyoblasts, myotomal myoblasts and myotubes. The intensity of alpha-sm actin expression decreased during the terminal steps of myogenesis and disappeared completely in mature cardiomyocytes and myofibres. Desmin was expressed in all prefusion myoblasts (type 1 and 2 myoblasts), myotubes, and in myofibres. The appearance of desmin in myoblasts of somites preceded by a few hours the expression of the alpha-actins (alpha-sr and alpha-sm). Our study on vimentin expression, limited to rat embryos, revealed that somite premyoblasts expressed only vimentin, type 1 myoblasts expressed vimentin and desmin, and type 2 myoblasts (rhabdomyoblasts) expressed desmin and alpha-actins (alpha-sr and alpha-sm). Our study demonstrates the resemblance between feto-embryonic myogenesis and myogenic neoplastic differentiation: desmin appears before the alpha-actins in embryonic myoblasts, and can be considered as a marker of an initial step in myogenic differentiation. alpha-sm actin, considered as a striated muscle cell feto-embryonic actin, is expressed transiently in skeletal myoblasts and cardiomyoblasts during development and reappears during neoplastic transformation of skeletal muscle.     

Myotubes differentiate optimally on substrates with tissue-like stiffness: pathological implications for soft or stiff microenvironments

Contractile myocytes provide a test of the hypothesis that cells sense their mechanical as well as molecular microenvironment, altering expression, organization, and/or morphology accordingly. Here, myoblasts were cultured on collagen strips attached to glass or polymer gels of varied elasticity. Subsequent fusion into myotubes occurs independent of substrate flexibility. However, myosin/actin striations emerge later only on gels with stiffness typical of normal muscle (passive Young's modulus, E approximately 12 kPa). On glass and much softer or stiffer gels, including gels emulating stiff dystrophic muscle, cells do not striate. In addition, myotubes grown on top of a compliant bottom layer of glass-attached myotubes (but not softer fibroblasts) will striate, whereas the bottom cells will only assemble stress fibers and vinculin-rich adhesions. Unlike sarcomere formation, adhesion strength increases monotonically versus substrate stiffness with strongest adhesion on glass. These findings have major implications for in vivo introduction of stem cells into diseased or damaged striated muscle of altered mechanical composition.     

Effects of mechanical stretching on caspase and IGF-1 expression during the proliferation process of myoblasts

It has been reported that the synthesis, degradation, and metabolism of muscle proteins in myoblasts, as well as the proliferation and differentiation of cells, are influenced by various related to extracellular signaling molecules, such as neural transmitters, growth factors, and hormones, when muscle tissue has been exposed to mechanical stimulation. However, reports regarding the expression of growth factors during mechanical stimulation of myoblasts are few, and many questions remain unanswered. We examined the mRNA expression of insulin-like growth factor 1 (IGF-1) in myoblasts subjected to mechanical stretching in vitro. In addition, apoptosis caused by intracellular stress has been reported to occur during muscle development at the embryonic stage. To clarify the expression of intracellular stress factors, we here investigated related gene expression. Expression of IGF-1 increased in the early stage of cell stretching, followed by a decrease in the late stage. This suggests that mechanical stimulation resulted in an immediate increase in IGF-1 expression, followed by a decrease as cells acclimated to the inducing environment. Caspase was significantly expressed in a stretch group at 12 hours after the beginning of mechanical stimulation, compared with a control group. This suggests that cellular proliferation is also regulated by intracellular stress factors involving the endoplasmic reticulum, mitochondria, and other organelles during the process of muscle proliferation and differentiation.     

Targeted and stable gene delivery into muscle cells by a two-step transfer system

We developed a muscle-specific gene delivery system based on two-step gene transfer. The first step involved adenovirus-mediated transfer of the ecotropic retrovirus receptor (EcoRec) gene driven by the muscle-specific desmin promoter. Both human primary myoblasts and fibroblasts were efficiently transduced with this adenovirus vector. However, expression of EcoRec was detected only in myoblasts. In the second step, EcoRec-expressing myoblasts could be stably transduced with the ecotropic retroviral vector with the beta-galactosidase gene. Approximately 15% of myoblasts were transduced by this two-step strategy. When the transduced myoblasts were differentiated into myotubes, extensive cell-cell fusion occurred, and the apparent number of beta-galactosidase-positive cells increased to 28%. These results indicate that our two-step gene delivery system could be used for targeted and stable gene transfer into muscle cells.     

Skeletal muscle fibrosis develops in response to desmin deletion

Skeletal muscle is a dynamic composite of proteins that responds to both internal and external cues to facilitate muscle adaptation. In cases of disease or altered use, these messages can be distorted resulting in myopathic conditions such as fibrosis. In this work, we describe a mild and progressive fibrotic adaptation in skeletal muscle lacking the cytoskeletal intermediate filament protein desmin. Muscles lacking desmin become progressively stiffer, accumulate increased collagen, and increase expression of genes involved in extracellular matrix turnover. Additionally, in the absence of desmin, skeletal muscle is in an increased state of inflammation and regeneration as indicated by increased centrally nucleated fibers, elevated inflammation and regeneration related gene expression, and increased numbers of inflammatory cells. These data suggest a potential link between increased cellular damage and the development of fibrosis in muscles lacking the cytoskeletal support of the desmin filament network.     

Some distinctive features of zebrafish myogenesis based on unexpected distributions of the muscle cytoskeletal proteins actin,myosin, desmin,alpha-actinin,troponin and titin

The current myofibrillogenesis model is based mostly on in vitro cell cultures and on avian and mammalian embryos in situ. We followed the expression of actin, myosin, desmin, alpha-actinin, titin, and troponin using immunofluorescence microscopy of zebrafish (Danio rerio) embryos. We could see young mononucleated myoblasts with sharp striations. The striations were positive for all the sarcomeric proteins. Desmin distribution during muscle maturation changes from dispersed aggregates to a perinuclear concentration to striated afterwards. We could not observe desmin-positive, myofibrillar-proteins-negative cells, and we could not find any non-striated distribution of sarcomeric proteins, such as stress fiber-like structures. Some steps, like fusion before striation, seem to be different in the zebrafish when compared with the previously described myogenesis sequences.     

Desmin/vimentin intermediate filaments are dispensable for many aspects of myogenesis

An expression vector was prepared containing a cDNA coding for a truncated version of the intermediate filament (IF) protein desmin. The encoded truncated desmin protein lacks a portion of the highly conserved alpha-helical rod region as well as the entire nonhelical carboxy-terminal domain. When transiently expressed in primary fibroblasts, or in differentiating postmitotic myoblasts and multinucleated myotubes, the truncated protein induces the complete dismantling of the preexisting vimentin or desmin/vimentin IF networks, respectively. Instead, in both cell types vimentin and desmin are packaged into hybrid spheroid bodies scattered throughout the cytoplasm. Despite the complete lack of intact IFs, myoblasts and myotubes expressing truncated desmin assemble and laterally align normal striated myofibrils and contract spontaneously in a manner indistinguishable from that of control myogenic cells. In older cultures the spheroid bodies shift from a longitudinal to a predominantly transverse orientation and loosely align along the I-Z-I-regions of striated myofibrils (Bennett, G.S., S. Fellini, Y. Toyama, and H. Holtzer. 1979. J. Cell Biol. 82:577-584), analogous to the translocation of intact desmin/vimentin IFs in control muscle. These results suggest the need for a critical reexamination of currently held concepts regarding the functions of desmin IFs during myogenesis.     

A Highlights from MBoC Selection: Nebulin binding impedes mutant desmin filament assembly

Desmin intermediate filaments (DIFs) form an intricate meshwork that organizes myofibers within striated muscle cells. The mechanisms that regulate the association of desmin to sarcomeres and their role in desminopathy are incompletely understood. Here we compare the effect nebulin binding has on the assembly kinetics of desmin and three desminopathy-causing mutant desmin variants carrying mutations in the head, rod, or tail domains of desmin (S46F, E245D, and T453I). These mutants were chosen because the mutated residues are located within the nebulin-binding regions of desmin. We discovered that, although nebulin M160–164 bound to both desmin tetrameric complexes and mature filaments, all three mutants exhibited significantly delayed filament assembly kinetics when bound to nebulin. Correspondingly, all three mutants displayed enhanced binding affinities and capacities for nebulin relative to wild-type desmin. Electron micrographs showed that nebulin associates with elongated normal and mutant DIFs assembled in vitro. Moreover, we measured significantly delayed dynamics for the mutant desmin E245D relative to wild-type desmin in fluorescence recovery after photobleaching in live-cell imaging experiments. We propose a mechanism by which mutant desmin slows desmin remodeling in myocytes by retaining nebulin near the Z-discs. On the basis of these data, we suggest that for some filament-forming desmin mutants, the molecular etiology of desminopathy results from subtle deficiencies in their association with nebulin, a major actin-binding filament protein of striated muscle.     

Talin2 is induced during striated muscle differentiation and is targeted to stable adhesion complexes in mature muscle

The cytoskeletal protein talin serves as an essential link between integrins and the actin cytoskeleton in several similar, but functionally distinct, adhesion complexes, including focal adhesions, costameres, and intercalated disks. Vertebrates contain two talin genes, TLN1 and TLN2, but the different roles of Talin1 and Talin2 in cell adhesion are unclear. In this report we have analyzed Talin1 and Talin2 in striated muscle. Using isoform-specific antibodies, we found that Talin2 is highly expressed in mature striated muscle. Using mouse C2C12 cells and primary human skeletal muscle myoblasts as models of muscle differentiation, we show that Talin1 is expressed in undifferentiated myoblasts and that Talin2 expression is upregulated during muscle differentiation at both the mRNA and protein levels. We have also identified regulatory sequences that may be responsible for the differential expression of Talin1 and Talin2. Using GFP-tagged Talin1 and Talin2 constructs, we found that GFP-Talin1 targets to focal adhesions while GFP-Talin2 targets to abnormally large adhesions in myoblasts. We also found that ectopic expression of Talin2 in myoblasts, which do not contain appreciable levels of Talin2, dysregulates the actin cytoskeleton. Finally we demonstrate that Talin2, but not Talin1, localizes to costameres and intercalated disks, which are stable adhesions required for the assembly of mature striated muscle. Our results suggest that Talin1 is the primary link between integrins and actin in dynamic focal adhesions in undifferentiated, motile cells, but that Talin2 may serve as the link between integrins and the sarcomeric cytoskeletonin stable adhesion complexes in mature striated muscle.     

Increased mechanosensitivity and nuclear stiffness in Hutchinson-Gilford progeria cells: effects of farnesyltransferase inhibitors

Hutchinson-Gilford progeria syndrome (HGPS), reportedly a model for normal aging, is a genetic disorder in children marked by dramatic signs suggestive for premature aging. It is usually caused by de novo mutations in the nuclear envelope protein lamin A. Lamins are essential to maintaining nuclear integrity, and loss of lamin A/C results in increased cellular sensitivity to mechanical strain and defective mechanotransduction signaling. Since increased mechanical sensitivity in vascular cells could contribute to loss of smooth muscle cells and the development of arteriosclerosis--the leading cause of death in HGPS patients--we investigated the effect of mechanical stress on cells from HGPS patients. We found that skin fibroblasts from HGPS patients developed progressively stiffer nuclei with increasing passage number. Importantly, fibroblasts from HGPS patients had decreased viability and increased apoptosis under repetitive mechanical strain, as well as attenuated wound healing, and these defects preceded changes in nuclear stiffness. Treating fibroblasts with farnesyltransferase inhibitors restored nuclear stiffness in HGPS cells and accelerated the wound healing response in HGPS and healthy control cells by increasing the directional persistence of migrating cells. However, farnesyltransferase inhibitors did not improve cellular sensitivity to mechanical strain. These data suggest that increased mechanical sensitivity in HGPS cells is unrelated to changes in nuclear stiffness and that increased biomechanical sensitivity could provide a potential mechanism for the progressive loss of vascular smooth muscle cells under physiological strain in HGPS patients.     

Stimulation of mechano-growth factor expression by myofibrillar proteins in murine myoblasts and myotubes

Mechano-growth factor (MGF) is a product of alternative splicing of the insulin-like growth factor 1 (IGF-1) mRNA. MGF is known to stimulate myoblast proliferation and to protect neurons and cardiomyocytes from apoptosis. MGF expression is dramatically increased in response to mechanical stimuli and tissue damage. The mechanisms of induction of MGF expression are as yet imperfectly understood. There is certain evidence that some protein factors able to stimulate MGF synthesis in normal myoblasts are released from damaged muscle. This study was undertaken to explore the nature of these protein inductors of MGF expression and to investigate the mechanism of their action. We report here that myofibrillar fraction of skeletal muscle homogenate activated MGF expression in murine myoblasts and myotubes in culture. The expression of another splice form of IGF-1 gene, IGF-1Ea, was also stimulated by myofibrils. Three myofibrillar proteins able to stimulate MGF synthesis were isolated. These proteins were identified by MALDI and immunoblotting as myomesin, myosin-binding protein C, and titin. The activation of MGF expression was associated with the increase of cAMP level in the cells. Inhibitor of adenylyl cyclase dideoxyadenosine arrested stimulation of MGF synthesis by all three myofibrillar proteins.     

Analysis of skeletal muscle function in the C57BL6/SV129 syncoilin knockout mouse

Syncoilin is a 64-kDa intermediate filament protein expressed in skeletal muscle and enriched at the perinucleus, sarcolemma, and myotendinous and neuromuscular junctions. Due to its pattern of cellular localization and binding partners, syncoilin is an ideal candidate to be both an important structural component of myocytes and a potential mediator of inherited myopathies. Here we present a report of a knockout mouse model for syncoilin and the results of an investigation into the effect of a syncoilin null state on striated muscle function in 6-8-week-old mice. An analysis of proteins known to associate with syncoilin showed that ablation of syncoilin had no effect on absolute expression or spatial localization of desmin or alpha dystrobrevin. Our syncoilin-null animal exhibited no differences in cardiotoxin-induced muscle regeneration, voluntary wheel running, or enforced treadmill exercise capacity, relative to wild-type controls. Finally, a mechanical investigation of isolated soleus and extensor digitorum longus indicated a potential differential reduction in muscle strength and resilience. We are the first to present data identifying an increased susceptibility to muscle damage in response to an extended forced exercise regime in syncoilin-deficient muscle. This study establishes a second viable syncoilin knockout model and highlights the importance of further investigations to determine the role of syncoilin in skeletal muscle.     

Role of cell division in differentiation of myoblasts infected with a temperature-sensitive mutant of Rous sarcoma virus.

The relationship between a potential requirement for cell DNA synthesis and the expression of differentiated muscle cell functions was investigated using primary chicken embryo myoblasts infected with a temperature-sensitive mutant of Rous sarcoma virus (RSV). Under optimized conditions, transformed myoblasts growing at the permissive temperature could differentiate into multinucleated myotubes, express muscle-specific myosin, desmin and acetylcholine receptors in the absence of DNA synthesis and cell division following a shift to the non-permissive temperature. Furthermore, the experiments demonstrate that individual RSV-infected myoblasts have two options: either to divide and express the transformed phenotype or to withdraw from the cell cycle and differentiate into myotubes. The choice between these options appears to depend on the protein-kinase activity of pp60src, the src gene product.     

3-D in vitro model of early skeletal muscle development

An understanding of the mechanical and mechano-molecular responses that occur during the differentiation of mouse C2C12 [corrected] myoblasts in 3-D culture is critical for understanding growth, which is important for progress towards producing a tissue-engineered muscle construct. We have established the main differences in force generation between skeletal myoblasts, dermal fibroblasts, and smooth muscle cells in a 3-D culture model in which cells contract a collagen gel construct. This model was developed to provide a reproducible 3-D muscle organoid in which differences in force generation could be measured, as the skeletal myoblasts fused to form myotubes within a collagen gel. Maintenance of the 3-D culture under sustained uni-axial tension, was found to promote fusion of myoblasts to form aligned multi-nucleate myotubes. Gene expression of both Insulin Like Growth Factor (IGF-1 Ea) and an isoform of IGF-1 Ea, Mechano-growth factor (IGF-1 Eb, also termed MGF), was monitored in this differentiating collagen construct over the time course of fusion and maturation (0-7 days). This identified a transient surge in both IGF-1 and MGF expression on day 3 of the developing construct. This peak of IGF-1 and MGF expression, just prior to differentiation, was consistent with the idea that IGF-1 stimulates differentiation through a Myogenin pathway [Florini et al., 1991: Mol. Endocrinol. 5:718-724]. MGF gene expression was increased 77-fold on day 3, compared to a 36-fold increase with IGF-1 on day 3. This indicates an important role for MGF in either differentiation or, more likely, a response to mechanical or tensional cues.     

Skeletal muscle cells express the profibrotic cytokine connective tissue growth factor (CTGF/CCN2), which induces their dedifferentiation

Fibrotic disorders are typified by excessive connective tissue and extracellular matrix (ECM) deposition that precludes normal healing processes of different tissues. Connective tissue growth factor (CTGF) seems to be involved in the fibrotic response. Several muscular dystrophies are characterized by a progressive weakness and wasting of the musculature, and by extensive fibrosis. However, the exact role of CTGF in skeletal muscle is unknown. Here we show that myoblasts and myotubes are able to synthesize CTGF in response to transforming growth factor type-beta (TGF-beta) and lysophosphatidic acid (LPA). CTGF induced several ECM constituents such as fibronectin, collagen type I and alpha4, 5, 6, and beta1 integrin subunits in myoblasts and myotubes. CTGF had an important inhibitory effect on muscle differentiation evaluated by the decrease in the nuclear translocation of the early muscle regulatory factor myogenin and myosin. Remarkable, CTGF treatment of myoblasts induced their dedifferentiation, characterized by down regulating MyoD and desmin, two markers of committed myoblasts, together with a strong reorganization of cytoskeletal filaments. These results provide novel evidence for the underlying mechanisms and participation of skeletal muscle cells in the synthesis and role of CTGF inducing fibrosis, inhibiting myogenesis and dedifferentiating myoblasts.     

Metavinculin: New insights into functional properties of a muscle adhesion protein

Metavinculin is a muscle-specific splice variant of the ubiquitously expressed cytoskeletal adaptor protein vinculin. Both proteins are thought to be co-expressed in all muscle types where they co-localize to microfilament-associated adhesion sites. It has been shown that a metavinculin-specific insertion of 68 amino acids alters the biochemical properties of the five-helix bundle in the tail domain. Here, we demonstrate that the metavinculin-specific helix H1′ plays an important role for protein stability of the tail domain, since a point mutation in this helix, R975W, which is associated with the occurrence of dilated cardiomyopathy in man, further decreases thermal stability of the metavinculin tail domain. In striated muscle progenitor cells (myoblasts), both, metavinculin and the R975W mutant show significantly reduced, albeit distinctive residency and exchange rates in adhesion sites as compared to vinculin. In contrast to previous studies, we show that metavinculin is localized in a muscle fiber type-dependent fashion to the costameres of striated muscle, reflecting the individual metabolic and physiological status of a given muscle fiber. Metavinculin expression is highest in fast, glycolytic muscle fibers and virtually absent in M. diaphragmaticus, a skeletal muscle entirely lacking fast, glycolytic fibers. In summary, our data suggest that metavinculin enrichment in attachment sites of muscle cells leads to higher mechanical stability of adhesion complexes allowing for greater shear force resistance.