Changes in the Mechanical Properties of Fast and Slow Skeletal Muscle after 7 and 21 Days of Restricted Activity in Rats.

Disuse muscle atrophy is usually accompanied by changes in skeletal muscle structure, signaling, and contractile potential. Different models of muscle unloading can provide valuable information, but the protocols of experiments with complete immobilization are not physiologically representative of a sedentary lifestyle, which is highly prevalent among humans now. In the current study, we investigated the potential effects of restricted activity on the mechanical characteristics of rat postural (soleus) and locomotor (extensor digitorum longus, EDL) muscles. The restricted-activity rats were kept in small Plexiglas cages (17.0 × 9.6 × 13.0 cm) for 7 and 21 days. After this, soleus and EDL muscles were collected for ex vivo mechanical measurements and biochemical analysis. We demonstrated that while a 21-day movement restriction affected the weight of both muscles, in soleus muscle we observed a greater decrease. The maximum isometric force and passive tension in both muscles also significantly changed after 21 days of movement restriction, along with a decrease in the level of collagen 1 and 3 mRNA expression. Furthermore, the collagen content itself changed only in soleus after 7 and 21 days of movement restriction. With regard to cytoskeletal proteins, in our experiment we observed a significant decrease in telethonin in soleus, and a similar decrease in desmin and telethonin in EDL. We also observed a shift towards fast-type myosin heavy chain expression in soleus, but not in EDL. In summary, in this study we showed that movement restriction leads to profound specific changes in the mechanical properties of fast and slow skeletal muscles. Future studies may include evaluation of signaling mechanisms regulating the synthesis, degradation, and mRNA expression of the extracellular matrix and scaffold proteins of myofibers.

Nonspecific Amyloid Aggregation of Chicken Smooth-Muscle Titin: In Vitro Investigations.

A giant multidomain protein of striated and smooth vertebrate muscles, titin, consists of tandems of immunoglobulin (Ig)- and fibronectin type III (FnIII)-like domains representing ß-sandwiches, as well as of disordered segments. Chicken smooth muscles express several titin isoforms of ~500-1500 kDa. Using various structural-analysis methods, we investigated in vitro nonspecific amyloid aggregation of the high-molecular-weight isoform of chicken smooth-muscle titin (SMTHMW, ~1500 kDa). As confirmed by X-ray diffraction analysis, under near-physiological conditions, the protein formed amorphous amyloid aggregates with a quaternary cross-ß structure within a relatively short time (~60 min). As shown by circular dichroism and Fourier-transform infrared spectroscopy, the quaternary cross-ß structure-unlike other amyloidogenic proteins-formed without changes in the SMTHMW secondary structure. SMTHMW aggregates partially disaggregated upon increasing the ionic strength above the physiological level. Based on the data obtained, it is not the complete protein but its particular domains/segments that are likely involved in the formation of intermolecular interactions during SMTHMW amyloid aggregation. The discovered properties of titin position this protein as an object of interest for studying amyloid aggregation in vitro and expanding our views of the fundamentals of amyloidogenesis.

NOS-dependent effects of plantar mechanical stimulation on mechanical characteristics and cytoskeletal proteins in rat soleus muscle during hindlimb suspension.

The study was aimed at investigating the mechanisms and structures which determine mechanical properties of skeletal muscles under gravitational unloading and plantar mechanical stimulation (PMS). We hypothesized that PMS would increase NO production and prevent an unloading-induced reduction in skeletal muscle passive stiffness. Wistar rats were hindlimb suspended and subjected to a daily PMS and one group of stimulated animals was also treated with nitric oxide synthase (NOS) inhibitor (L-NAME). Animals received mechanical stimulation of the feet for 4 h a day throughout 7-day hindlimb suspension (HS) according to a scheme that mimics the normal walking of the animal. Seven-day HS led to a significant reduction in soleus muscle weight by 25%. However, PMS did not prevent the atrophic effect induced by HS. Gravitational unloading led to a significant decrease in maximum isometric force and passive stiffness by 38% and 31%, respectively. The use of PMS prevented a decrease in the maximum isometric strength of the soleus muscle. At the same time, the passive stiffness of the soleus in the PMS group significantly exceeded the control values by 40%. L-NAME (NOS inhibitor) administration attenuated the effect of PMS on passive stiffness and maximum force of the soleus muscle. The content of the studied cytoskeletal proteins (α-actinin-2, α-actinin-3, desmin, titin, nebulin) decreased after 7-day HS, but this decrease was successfully prevented by PMS in a NOS-dependent manner. We also observed significant decreases in mRNA expression levels of α-actinin-2, desmin, and titin after HS, which was prevented by PMS. The study also revealed a significant NOS-dependent effect of PMS on the content of collagen-1a, but not collagen-3a. Thus, PMS during mechanical unloading is able to maintain soleus muscle passive tension and force as well as mRNA transcription and protein contents of cytoskeletal proteins in a NOS-dependent manner.

Amyloid Aggregates of Smooth-Muscle Titin Impair Cell Adhesion.

Various amyloid aggregates, in particular, aggregates of amyloid ß-proteins, demonstrate in vitro and in vivo cytotoxic effects associated with impairment of cell adhesion. We investigated the effect of amyloid aggregates of smooth-muscle titin on smooth-muscle-cell cultures. The aggregates were shown to impair cell adhesion, which was accompanied by disorganization of the actin cytoskeleton, formation of filopodia, lamellipodia, and stress fibers. Cells died after a 72-h contact with the amyloid aggregates. To understand the causes of impairment, we studied the effect of the microtopology of a titin-amyloid-aggregate-coated surface on fibroblast adhesion by atomic force microscopy. The calculated surface roughness values varied from 2.7 to 4.9 nm, which can be a cause of highly antiadhesive properties of this surface. As all amyloids have the similar structure and properties, it is quite likely that the antiadhesive effect is also intrinsic to amyloid aggregates of other proteins. These results are important for understanding the mechanisms of the negative effect of amyloids on cell adhesion.

Myosin Binding Protein-C Forms Amyloid-Like Aggregates In Vitro.

This work investigated in vitro aggregation and amyloid properties of skeletal myosin binding protein-C (sMyBP-C) interacting in vivo with proteins of thick and thin filaments in the sarcomeric A-disc. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) found a rapid (5-10 min) formation of large (>2 µm) aggregates. sMyBP-C oligomers formed both at the initial 5-10 min and after 16 h of aggregation. Small angle X-ray scattering (SAXS) and DLS revealed sMyBP-C oligomers to consist of 7-10 monomers. TEM and atomic force microscopy (AFM) showed sMyBP-C to form amorphous aggregates (and, to a lesser degree, fibrillar structures) exhibiting no toxicity on cell culture. X-ray diffraction of sMyBP-C aggregates registered reflections attributed to a cross-ß quaternary structure. Circular dichroism (CD) showed the formation of the amyloid-like structure to occur without changes in the sMyBP-C secondary structure. The obtained results indicating a high in vitro aggregability of sMyBP-C are, apparently, a consequence of structural features of the domain organization of proteins of this family. Formation of pathological amyloid or amyloid-like sMyBP-C aggregates in vivo is little probable due to amino-acid sequence low identity (<26%), alternating ordered/disordered regions in the protein molecule, and S-S bonds providing for general stability.

Study of the complement activation by amyloid aggregates of smooth muscle titin in vitro.

The giant muscle protein, titin, is the third most abundant protein in muscle (after myosin and actin). It was shown previously that smooth muscle titin (SMT) with a molecular mass of 500 kDa can form in vitro amorphous amyloid aggregates in two conditions: in solution of low ionic strength (0.15 M Glycine-KOH, pH 7.0) (SMT(Gly) aggregates) and in solution with ionic strength in the physiological range (0.2 M KCl, 20 mM imidazole, pH 7.2-7.4) (SMT(KCl) aggregates). Such aggregation in vivo, which may play a pathological or functional role, is not excluded. In view of the fact that some pathological amyloids can activate the classical and alternative pathways of complement system, we investigated the binding of complement component C1q and C3b to smooth muscle titin amyloid aggregates. The binding of С1q and C3b to SMT aggregates was not observed with ELISA assay. Since SMT aggregates do not activate the complement system, they are hardly implicated in the inflammatory process caused by muscle damage in amyloidoses.Abbreviations: SMT: smooth muscle titin; SMT(KCl) aggregates: SMT aggregates in solution containing 0.2 M KCl, 10 mM imidazole, pH 7.0; SMT(Gly) aggregates: SMT aggregates in solution containing 0.15 M glycine-KOH, pH 7.2-7.4; MAC: membrane attack complex; DLS: dynamic light scattering; NHS: Normal Human Serum.

New Model for Stacking Monomers in Filamentous Actin from Skeletal Muscles of Oryctolagus cuniculus.

To date, some scientific evidence (limited proteolysis, mass spectrometry analysis, electron microscopy (EM)) has accumulated, which indicates that the generally accepted model of double-stranded of filamentous actin (F-actin) organization in eukaryotic cells is not the only one. This entails an ambiguous understanding of many of the key cellular processes in which F-actin is involved. For a detailed understanding of the mechanism of F-actin assembly and actin interaction with its partners, it is necessary to take into account the polymorphism of the structural organization of F-actin at the molecular level. Using electron microscopy, limited proteolysis, mass spectrometry, X-ray diffraction, and structural modeling we demonstrated that F-actin presented in the EM images has no double-stranded organization, the regions of protease resistance are accessible for action of proteases in F-actin models. Based on all data, a new spatial model of filamentous actin is proposed, and the F-actin polymorphism is discussed.

Cellular and molecular signatures of alcohol-induced myopathy in women.

Alcoholic myopathy is characterized by the reduction in cross-sectional area (CSA) of muscle fibers and impaired anabolic signaling. The goal of the current study was to investigate the causes and compare the changes in CSA and fiber type composition with the modifications of anabolic and catabolic signaling pathways at the early stages of chronic alcohol consumption in women. Skeletal muscle samples from 5 female patients with alcohol abuse (AL; 43 ± 5 yr old; alcohol abuse duration 5,6 ± 0,6 yr) were compared with the muscle from the control group of 8 healthy women (C; 35 ± 4 yr old). The average daily dose of alcohol consumption was 110 ± 10 ml of pure ethanol. In women patients, a significant decrease in CSA of type I and II muscle fibers, titin and nebulin content, plasma IGF-1 level and total IRS-1, p-Akt and p-4E-BP1 in vastus lateralis was found in comparison with the control group. The p-AMPK level was found to be increased versus the control group. In women patients with chronic alcoholic myopathy 1) both fast and slow muscle fibers are subjected to atrophy; 2) impairments in IGF-I-dependent signaling and pathways controlling translation initiation (AMPK/mTOR/4E-BP1), but not translation elongation, are observed; 3) the level of calpain-1 and ubiquitinated proteins increases, unlike E3 ligases content.

Increased Autolysis of µ-Calpain in Skeletal Muscles of Chronic Alcohol-Fed Rats.

BACKGROUND: Proteolysis can proceed via several distinct pathways such as the lysosomal, calcium-dependent, and ubiquitin-proteasome-dependent pathways. Calpains are the main proteases that cleave a large variety of proteins, including the giant sarcomeric proteins, titin and nebulin. Chronic ethanol feeding for 6 weeks did not affect the activities of µ-calpain and m-calpain in the m. gastrocnemius. In our research, changes in µ-calpain activity were studied in the m. gastrocnemius and m. soleus of chronically alcohol-fed rats after 6 months of alcohol intake. METHODS: SDS-PAGE analysis was applied to detect changes in titin and nebulin contents. Titin phosphorylation analysis was performed using the fluorescent dye Pro-Q Diamond. Western blotting was used to determine µ-calpain autolysis as well as µ-calpain and calpastatin contents. The titin and nebulin mRNA levels were assessed by real-time PCR. RESULTS: The amounts of the autolysed isoform (78 kDa) of full-length µ-calpain (80 kDa) increased in the m. gastrocnemius and m. soleus of alcohol-fed rats. The calpastatin content increased in m. gastrocnemius. Decreased intact titin-1 (T1) and increased T2-proteolytic fragment contents were found in the m. gastrocnemius and m. soleus of the alcohol-fed rats. The nebulin content decreased in the rat gastrocnemius muscle of the alcohol-fed group. The phosphorylation levels of T1 and T2 were increased in the m. gastrocnemius and m. soleus, and decreased titin and nebulin mRNA levels were observed in the m. gastrocnemius. The nebulin mRNA level was increased in the soleus muscle of the alcohol-fed rats. CONCLUSIONS: In summary, our data suggest that prolonged chronic alcohol consumption for 6 months resulted in increased autolysis of µ-calpain in rat skeletal muscles. These changes were accompanied by reduced titin and nebulin contents, titin hyperphosphorylation, and development of hindlimb muscle atrophy in the alcohol-fed rats.

Amyloids: The History of Toxicity and Functionality.

Proteins can perform their specific function due to their molecular structure. Partial or complete unfolding of the polypeptide chain may lead to the misfolding and aggregation of proteins in turn, resulting in the formation of different structures such as amyloid aggregates. Amyloids are rigid protein aggregates with the cross-ß structure, resistant to most solvents and proteases. Because of their resistance to proteolysis, amyloid aggregates formed in the organism accumulate in tissues, promoting the development of various diseases called amyloidosis, for instance Alzheimer's diseases (AD). According to the main hypothesis, it is considered that the cause of AD is the formation and accumulation of amyloid plaques of Aß. That is why Aß-amyloid is the most studied representative of amyloids. Therefore, in this review, special attention is paid to the history of Aß-amyloid toxicity. We note the main problems with anti-amyloid therapy and write about new views on amyloids that can play positive roles in the different organisms including humans.

Congo Red and amyloids: history and relationship.

Staining with Congo Red (CR) is a qualitative method used for the identification of amyloids in vitro and in tissue sections. However, the drawbacks and artefacts obtained when using this dye can be found both in vitro and in vivo Analysis of scientific data from previous studies shows that CR staining alone is not sufficient for confirmation of the amyloid nature of protein aggregates in vitro or for diagnosis of amyloidosis in tissue sections. In the present paper, we describe the characteristics and limitations of other methods used for amyloid studies. Our historical review on the use of CR staining for amyloid studies may provide insight into the pitfalls and caveats related to this technique for researchers considering using this dye.

Anti-amyloid activities of three different types of water-soluble fullerene derivatives.

Anti-amyloid activity, aggregation behaviour, cytotoxicity and acute toxicity were investigated for three water-soluble fullerene derivatives with different types of solubilizing addends. All investigated compounds showed a strong anti-amyloid effect in vitrocaused by interaction of the water-soluble fullerene derivatives with the Ab(1-42)-peptide and followed by destruction of the amyloid fibrils. Notably, all of the studied fullerene derivatives showed very low cytotoxicity and low acute toxicity in mice (most promising compound 3 was more than four times less toxic than aspirin). Strong anti-amyloid effect of the fullerene derivatives together with low toxicity reveals high potential of these compounds as drug candidates for treatment of neurodegenerative diseases.

Different amyloid aggregation of smooth muscles titin in vitro.

A comparative study of amyloid properties of the aggregates of smooth muscle titin (SMT) from chicken gizzard was carried out. These aggregates were formed in two solutions: 0.15 M glycine-KOH, pH 7.2-7.4 (SMT(Gly)) and 0.2 M KCl, 10 mM imidazole, pH 7.0 (SMT(KCl)). Electron microscopy data showed that SMT aggregates has an amorphous structure in both cases. The results of atomic-force microscopy demonstrated slight differences in morphology in two types of aggregates. The SMT(Gly) aggregates were represented as branching chains, composed of spherical aggregates approximately 300-500 nm in diameter and up to 35 nm in height. The SMT(KCl) aggregates formed sponge-like structures with strands of 8-10 nm in height. Structural analysis of SMT aggregates by X-ray diffraction revealed the presence of cross-ß-sheet structure in the samples under study. In the presence of SMT(Gly) aggregates, thioflavine T fluorescence intensity was higher (~3-fold times) compared with that in the presence of SMT(KCl) aggregates. Congo red-stained SMT(Gly) aggregates had yellow to apple-green birefringence under polarized light, which was not observed for SMT(KCl) aggregates. Dynamic light scattering data showed the similar rate of aggregation for both types of aggregates, though SMT(KCl) aggregates were able to partially disaggregate under increased ionic strength of the solution. The ability of SMT to aggregation followed by disaggregation may be functionally significant in the cell.

Nuances of electrophoresis study of titin/connectin.

Almost 40 years has passed since the discovery of giant elastic protein titin (also known as connectin) of striated and smooth muscles using gel electrophoresis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis is a major technique for studying the isoform composition and content of titin. This review provides historical insights into the technical aspects of the electrophoresis methods used to identify titin and its isoforms. We particularly focus on the nuances of the technique that improve the preservation of its primary structure so that its high molecular weight isoforms can be visualized.

Smooth muscle titin forms in vitro amyloid aggregates.

Amyloids are insoluble fibrous protein aggregates, and their accumulation is associated with amyloidosis and many neurodegenerative diseases, including Alzheimer's disease. In the present study, we report that smooth muscle titin (SMT; 500 kDa) from chicken gizzard forms amyloid aggregates in vitro This conclusion is supported by EM data, fluorescence analysis using thioflavin T (ThT), Congo red (CR) spectroscopy and X-ray diffraction. Our dynamic light scattering (DLS) data show that titin forms in vitro amyloid aggregates with a hydrodynamic radius (Rh) of approximately 700-4500 nm. The initial titin aggregates with Rh approximately 700 nm were observed beyond first 20 min its aggregation that shows a high rate of amyloid formation by this protein. We also showed using confocal microscopy the cytotoxic effect of SMT amyloid aggregates on smooth muscle cells from bovine aorta. This effect involves the disorganization of the actin cytoskeleton and result is cell damage. Cumulatively, our results indicate that titin may be involved in generation of amyloidosis in smooth muscles.