The central role of YB-1 in messenger ribonucleoprotein particle (mRNP) metabolism and stress-granule biology highlights the importance of defining the determinants of its self-assembly. YB-1 fibrillogenesis has been attributed primarily to the cold shock domain (CSD). Here, we show that the YB-1 fragment spanning residues 1-129 (AP-CSD) form amyloid fibrils under near-physiological ionic strength (0.12-0.15 M KCl). Fibrillization proceeds without a pronounced exponential growth phase and increases approximately linearly over 45-50 h. Far-UV circular dichroism (CD) and attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) indicate no substantial change in overall secondary-structure content during aggregation. In parallel, 1H nuclear magnetic resonance (NMR) spectroscopy reveals the depletion of soluble species, and oriented fiber X-ray diffraction displays the hallmark cross-β reflections at approximately 4.7 Å and 10 Å. The prolonged formation time implies an activation barrier that is unlikely to require global refolding. Instead, it may reflect early association events such as dimerization or other local rearrangements required for primary nucleation, followed by consolidation into stable intermolecular contacts. Aggregation that preserves a largely native-like fold while establishing cross-β order may reduce recognition by cellular quality-control systems that preferentially target globally unfolded or strongly destabilized states. This provides a plausible framework for how YB-1 derived assemblies could persist under stress and during age-associated proteostasis decline.

The sarcomeric giant protein titin affects the passive elasticity of the heart muscle and is crucial for proper cardiac function, including diastolic relaxation of the left ventricle. A useful common method for studying titin is electrophoretic analysis which can be used to examine the distribution of its isoforms in the heart. There are 5 titin parameters that can be analyzed: the N2BA/N2B isoforms ratio, the T2/T1 bands ratio, Cronos isoform content, NT isoform content, the total titin-to-myosin heavy chain (TT/MHC) ratio. These parameters can only be assessed through electrophoresis of giant proteins. It is known that these parameters are related to various biomolecular processes in muscle cells, such as providing of elastic properties, turnover, contraction, and maintaining a highly ordered sarcomere structure. In this review, we discuss the diagnostic potential of electrophoretic visualization of cardiac titin changes in various human heart diseases and animal models of physiological adaptations or pathologies.

Renocardiac syndrome type 4 (RCS4) is a common comorbid pathology, but the mechanisms of kidney dysfunction-induced cardiac remodeling and the involvement of cardiac progenitor cells (CPCs) in this process remain unclear. The aim of this study was to investigate the structural and functional changes in the cardiac muscle in RCS4 induced by unilateral ureteral obstruction (UUO) and the role of nestin+ CPCs in these. Heart function and localization of nestin+ cells in the myocardium were assessed using nestin-GFP transgenic mice subjected to UUO for 14 and 28 days. UUO resulted in cardiac hypertrophy, accompanied by an elongation of the QRS wave on the ECG, decreased expression of Cxcl1, Cxcl9, and Il1b, reduced the number of CD11b+ cells, and increased in titin isoform parameters, such as T1/MHC and TT/MHC ratios, without changes in fibrosis markers. The number of nestin+ cells increased in the myocardium with increased duration of UUO and displayed an SCA-1+TBX5+ phenotype, consistent with CPCs. Thus, cardiac pathology in RCS4 was manifested by cardiomyocyte hypertrophy with changes in the electrophysiological phenotype of the heart, not accompanied by fibrosis or inflammation. Nestin+ cardiac cells retained the CPC phenotype during UUO, and their number increased, which suggests their participation in regenerative processes in the heart.

Seasonal changes in fatty acid composition in four skeletal muscles of the true hibernating Yakutian long-tailed ground squirrel Urocitellus undulatus were studied. Measurements were taken on animals of four experimental groups: summer active, autumn active, winter hibernating, and winter active. An increase in total fatty acids was found in winter in the quadriceps femoris muscle (m. vastus lateralis), triceps forearm muscle (m. triceps), and lumbar muscle (m. psoas). A decrease in the total content of saturated fatty acids in all muscles was observed during the winter period. The increase in the total content of monounsaturated fatty acids in winter hibernating animals occurred in the quadriceps femoris muscle, triceps forearm muscle, and lumbar muscle. In winter active animals, the total content of polyunsaturated fatty acids increased in quadriceps femoris muscle and lumbar muscle. A statistically significant decrease in the content of palmitic acid in hibernating and winter active animals compared to summer and autumn animals was found in all muscles studied. The content of palmitoleic acid increased in hibernating animals in the quadriceps femoris muscle and lumbar muscle. In the triceps forearm muscle, the content of palmitoleic acid was increased in autumn active and winter hibernating animals. The content of oleic acid was elevated in all muscles in winter hibernating animals relative to active autumn animals. Linoleic acid content was significantly increased in winter active animals in all muscles except the calf muscle. Dihomo-gamma-linolenic acid increased in all muscles during the autumn period with a decrease in content in winter hibernating and winter active animals to the level of summer (seasonal) controls. The results obtained indicate that most of the changes in fatty acid composition have the same direction in all four studied skeletal muscles of the long-tailed ground squirrel. The possible role of seasonal changes in fatty acid composition and fatty acid participation in biochemical processes in the muscle tissue of the long-tailed ground squirrel is discussed.

Protein association and aggregation are fundamental processes that play critical roles in a variety of biological phenomena from cell signaling to the development of incurable diseases, including amyloidoses. Understanding the basic biophysical principles governing protein aggregation processes is of crucial importance for developing treatment strategies for diseases associated with protein aggregation, including sarcopenia, as well as for the treatment of pathological processes associated with the disruption of functional protein complexes. This work, using a set of methods such as atomic force microscopy (AFM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction, as well as bioinformatics analysis, investigated the structures of complexes formed by titin and myosin-binding protein C (MyBP-C). TEM revealed the formation of morphologically ordered aggregates in the form of beads during co-incubation of titin and MyBP-C under close-to-physiological conditions (175 mM KCl, pH 7.0). AFM showed the formation of a relatively homogeneous film with local areas of relief change. Fluorimetry with thioflavin T, as well as FTIR spectroscopy, revealed signs of an amyloid-like structure, including a signal in the cross-β region. X-ray diffraction showed the presence of a cross-β structure characteristic of amyloid aggregates. Such structural features were not observed in the control samples of the investigated proteins separately. In sarcomeres, these proteins are associated with each other, and this interaction plays a partial role in the formation of a strong sarcomeric cytoskeleton. We found that under physiological ionic-strength conditions titin and MyBP-C form complexes in which an amyloid-like structure is present. The possible functional significance of amyloid-like aggregation of these proteins in muscle cells in vivo is discussed.

Abstract—Titin is a multidomain protein of striated and smooth muscles of vertebrates. The protein consists of repeating immunoglobulin-like (Ig) and fibronectin-like (FnIII) domains, which are β-sandwiches with a predominant β-structure, and also contains disordered regions. In this work, the methods of atomic force microscopy (AFM), X-ray diffraction, and Fourier transform infrared spectroscopy were used to study the morphology and structure of aggregates of rabbit skeletal muscle titin obtained in two different solutions: 0.15 M glycine-KOH, pH 7.0 and 200 mM KCl, 10 mM imidazole, pH 7.0. According to AFM data, skeletal muscle titin formed amorphous aggregates of different morphologies in the above two solutions. Amorphous aggregates of titin formed in a solution containing glycine consisted of much larger particles than aggregates of this protein formed in a solution containing KCl. The “KCl-aggregates” according to AFM data had the form of a “sponge”-like structure, while amorphous “glycine-aggregates” of titin formed “branching” structures. Spectrofluorometry revealed the ability of “glycine-aggregates” of titin to bind to the dye thioflavin T (TT), and X-ray diffraction revealed the presence of one of the elements of the amyloid cross β-structure, a reflection of 4.6 Å, in these aggregates. These data indicate that “glycine-aggregates” of titin are amyloid or amyloid-like. No similar structural features were found in “KCl-aggregates” of titin; they also did not show the ability to bind to thioflavin T, indicating the non-amyloid nature of these titin aggregates. Fourier transform infrared spectroscopy revealed differences in the secondary structure of the two types of titin aggregates. The data we obtained demonstrate the features of structural changes during the formation of intermolecular bonds between molecules of the giant titin protein during its aggregation. The data expand the understanding of the process of amyloid protein aggregation.

Using produced polyclonal antibodies specific to the N-terminal sequence (residues 61-298) of rat obscurin, we investigated the isoform composition of this protein in 4 striated muscles: myocardium of the left ventricle, diaphragm, skeletal m. gastrocnemius (containing mainly fast fibers), and m. soleus (containing mainly slow fibers). The m. gastrocnemius, m. soleus, and diaphragm were found to have 2 giant isoforms of obscurin: a smaller A-isoform and a larger B-isoform. Their molecular weights were ~870 and ~1150 kDa in the diaphragm and m. gastrocnemius and ~880 and ~1130 kDa in m. soleus, respectively. The B-isoform to A-isoform ratio was 1:3 in the diaphragm and m. soleus and 1:4 in the m. gastrocnemius. In the left-ventricular myocardium, A-isoform of obscurin with a molecular weight of ~880 kDa was found. No other obscurin isoforms or their fragments within the molecular weight range of 10 up to ~800 kDa were revealed in the investigated rat striated muscles. The antibodies produced are recommended for research into qualitative and quantitative changes of giant obscurin isoforms in rat striated muscles in the norm and during the development of pathological processes.

The review provides a brief analysis of the current knowledge of such a post-translational modification of titin as phosphorylation, with a focus on changes that occur during the development of heart diseases. Studies using animal models of heart diseases and cardiac biopsy from patients with various pathologies reveal changes in the level of titin phosphorylation compared to healthy controls. The development of cardiac pathology is typically accompanied by hyperphosphorylation of the S11878 site and hypophosphorylation of the S12022 site in the titin PEVK region, as well as changes in the level of site phosphorylation in the titin N2B region. The cooperative functional effect of these changes is an increase in the stiffness of cardiomyocytes and cardiac muscle as a whole, based on the viscoelastic properties of titin. Changes in the latter, in turn, result from hypo- or hyperphosphorylation of certain titin sites. The review also addresses a number of therapeutic approaches aimed at modifying titin phosphorylation levels as a means to manage viscoelastic properties of the pathological myocardium in order to normalize its contractility.

Small-angle X-ray scattering (SAXS) and Fourier transform infrared (FTIR) spectroscopy were used to investigate structural peculiarities of two types of amyloid aggregates of smooth muscle titin, which differed in their morphology and ability to disaggregate, and differently bound thioflavin T dye. SAXS showed that the structure/shape of the two titin aggregate types was close to a flat shape. FTIR spectroscopy revealed no differences in the secondary structure of the two types. These data suggest that both types of "flat-shape" titin aggregates are identical in their secondary structure and, as shown previously, have a quaternary cross-β structure. An assumption was made that the most stable supramolecular complexes of a cross-β structure, which do not differ in their secondary structure, formed first during the aggregation of smooth muscle titin. Then, depending on ambient conditions, these supramolecular structures could form titin aggregates of different morphology and properties.

The process of amyloid aggregation is quite complex and poorly studied. In this paper, summarizing the previously obtained results on the aggregation of the multidomain smooth muscle protein titin, we tried to complement the idea of its amyloid aggregation by presenting a new, in our opinion, possible mechanism. The main conclusion is that the ability of titin to form amorphous aggregates seems to be the only possible means of aggregation of this protein. Apparently, only individual sections of the molecules, and not the entire protein, are involved in the formation of the amyloid structure in amorphous aggregates of smooth muscle titin. This feature distinguishes titin from other amyloid or amyloid-like proteins due to the large size of the molecule. The possible energy landscape underlying the formation of amyloid aggregates of titin is discussed.

Protein oligomers are important intermediates in the formation of amyloid fibrils. In amyloidosis, for example, Alzheimer’s disease, oligomers can have a toxic effect on cells. This paper describes the distinctive features of oligomerization of multidomain muscle proteins, smooth muscle titin, and myosin-binding protein C (C-protein) of skeletal muscles consisting of FnIII-like and IgC2-like domains and capable of forming amyloid amorphous aggregates in vitro. Under conditions of low ionic strength (below physiological values), the C-protein formed stable oligomers that were not involved in further aggregation. Smooth muscle titin formed oligomers under conditions of high ionic strength (μ 0.6), which were precursors of amyloid amorphous aggregates of this protein.The results we obtained expand the understanding of the process of protein aggregation.

The process of amyloid aggregation is quite complex and poorly understood. In this work, having summarized previously obtained results on the aggregation of the multidomain smooth muscle protein titin, an attempt has been made to expand understanding of this process, and a new possible mechanism by which amyloid aggregation of titin may occur is delineated. Our main conclusion is that the ability of titin to form amorphous aggregates seems to be the only possible way of aggregation of this protein. Most likely, only separate parts of the molecules, but not the whole protein, are involved in the formation of the amyloid structure in amorphous aggregates of smooth muscle titin. This feature, given the large size of the protein molecule, distinguishes titin from other amyloid or amyloid-like proteins. The paper discusses the potential energy landscape underlying the formation of titin amyloid aggregates.

Changes in the content of heat shock protein 90 (HSP90) in m. soleus (contains mainly fibers expressing the “slow” isoform I MyHC) and m. gastrocnemius (contains mainly fibers expressing the “fast” isoforms II MyHC) of a true hibernant, the long-tailed ground squirrel (Urocitellus undulatus), during different periods of the annual cycle, summer activity (seasonal control), hypothermia/winter torpor, and winter (interbout) activity, were studied. It was found that despite the development of atrophic changes that were more pronounced in the “fast” m. gastrocnemius, the content of HSP90 in both muscles did not change throughout the hibernation period. The role of HSP90 in maintaining the stability of the titin giant sarcomeric protein molecules during the periods of the animal’s entry into and exit from hypothermia, when the activity of calpain proteases increased due to the increased content of Ca2+ in the cytosol of muscle cells, as well as during hypothermia, when the activity of calpains most likely was not completely inhibited, was discussed. During the winter/interbout activity, when there was an increased titin turnover in the striated ground squirrel muscles, a constant content of HSP90 was apparently necessary for the correct folding of newly synthesized titin molecules and their embedding into sarcomeres, as well as for the removal of improperly folded and old titin molecules/fragments, as well as other proteins. Thus, HSP90 proteostasis in skeletal muscles of the long-tailed ground squirrel could contribute to maintaining a stable level of titin and, possibly, other sarcomeric proteins during hibernation, which, in turn, would contribute to maintaining a highly ordered sarcomeric structure and the necessary level of contractile muscle activity in different phases of the hibernation–wakefulness cycle.

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 β-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-β structure within a relatively short time (~60 min). As shown by circular dichroism and Fourier-transform infrared spectroscopy, the quaternary cross-β 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.

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.

In mammals, prolonged mechanical unloading results in a significant decrease in passive stiffness of postural muscles. The nature of this phenomenon remains unclear. The aim of the present study was to investigate possible causes for a reduction in rat soleus passive stiffness after 7 and 14 days of unloading (hindlimb suspension, HS). We hypothesized that HS-induced decrease in passive stiffness would be associated with calpain-dependent degradation of cytoskeletal proteins or a decrease in actomyosin interaction. Wistar rats were subjected to HS for 7 and 14 days with or without PD150606 (calpain inhibitor) treatment. Soleus muscles were subjected to biochemical analysis and ex vivo measurements of passive tension with or without blebbistatin treatment (an inhibitor of actomyosin interactions). Passive tension of isolated soleus muscle was significantly reduced after 7- and 14-day HS compared to the control values. PD150606 treatment during 7- and 14-day HS induced an increase in alpha-actinin-2 and -3, desmin contents compared to control, partly prevented a decrease in intact titin (T1) content, and prevented a decrease in soleus passive tension. Incubation of soleus muscle with blebbistatin did not affect HS-induced reductions in specific passive tension in soleus muscle. Our study suggests that calpain-dependent breakdown of cytoskeletal proteins, but not a change in actomyosin interaction, significantly contributes to unloading-induced reductions in intrinsic passive stiffness of rat soleus muscle.

Myofascial pain syndrome caused by damage to the paravertebral muscles is considered as one of the causes of chronic back pain. At the same time, there is not enough information about the condition of the paravertebral muscles, and it is contradictory. The aim of the work is to elucidate the presence and severity of structural and functional changes in the paravertebral muscles and their role in the development of chronic nonspecific pain in the lower back in women. Material and methods. Morphological, immunohistochemical examination of a muscle tissue biopsy was performed in 17 patients aged 24 to 59 years (average age — 41.5 ± 12.1 years) with CNS (average duration of pain syndrome 10.0 ± 6.9 months) caused by myofascial pain syndrome, as well as determination by gel electrophoresis of the isoform composition of giant sarcomeric proteins titin and nebulin. Results. Morphological examination revealed no signs of necrosis, proliferation of connective and adipose tissue, inflammatory infiltration. The transformation of the myosin phenotype in the direction of an increase in the proportion of “fast” type II muscle fibers was revealed in the biopsies of the patients’ muscles. A decrease in the content of giant titin and nebulin proteins associated with myosin and actin in the sarcomere was also found. Conclusion. The data obtained indicate a violation of the contractile function of the paravertebral muscle in CNS.

A feature of skeletal muscle X-protein aggregation is the formation of helical fibrils from twisted ribbons, which were previously observed by electron microscopy. To get a deeper understanding of polymorphism of X-protein aggregates, electron microscopy was used to study X-protein aggregation under various conditions. It was found that X-protein formed various types of aggregates: amorphous aggregates, protofibrils, bundles of linear fibrils, and helical fibrils formed from twisted ribbons, presumably of amyloid nature. In order to confirm this assumption, structural studies of X-protein helical fibrils assembled from twisted ribbons were carried out using data from X-ray diffraction. The analysis revealed the presence of strongly blurred reflections at 4.6 and 10 Å. These results indicate that the fibrils of X-protein do not possess a quaternary cross-β-structure that is characteristic for amyloid fibrils of known proteins and peptides, such as, for example, insulin, amyloid β-peptide and titin. These results contradict the previously obtained data on amyloid nature of the X-protein aggregates, the interpretation of which was based on the results on the binding of the “amyloid” dyes Congo Red and Thioflavin T to its fibrils. The ability of X-protein to form non-amyloid aggregates in vitro may be due to a defense mechanism developed through evolution that prevents the formation of toxic insoluble amyloid aggregates in cells.

Seasonal changes in mRNA levels of the giant sarcomeric cytoskeletal proteins titin and obscurin were studied in the skeletal m. longissimus dorsi and the cardiac (left ventricular) muscle in the long-tailed ground squirrel Urocitellus undulatus using real-time RT-PCR. The animals were divided into the following experimental groups: “summer activity”, “fall activity”, “hypothermia” (hibernation), “winter activity” (n = 5 per group). In the cardiac muscle of “hypothermia” animals, titin mRNA levels decreased by 28 0.01); in the other three groups, no statistically significant differences in this parameter were found. In m. longissimus dorsi of “hypothermia” and “winter activity” animals, titin mRNA levels increased by 2.9 (p ≤ 0.01) and 3.6 (p ≤ 0.01) times, respectively, with no statistically significant differences in this parameter in “summer activity” and “fall activity” animals. Obscurin mRNA levels increased by 3.4–3.6 times (p ≤ 0.01) in the cardiac muscle of “fall activity”, “hypothermia” and “winter activity” animals, and by 3.0 and 3.6 times (p ≤ 0.01) in the skeletal muscle of “hypothermia” and “winter activity” animals, respectively. Thus, we report here for the first time the data on the differential expression of titin and obscurin mRNAs, indicating concerted changes in the giant cytoskeletal proteins in muscles of the long-tailed ground squirrel during hibernation. The results are discussed in the context of striated muscle adaptation to hibernation in this rodent species.

Hibernation is used by specie as diverse as ground squirrels, snakes and bats to cope with reduced food availability and lowered temperatures and is a unique model for studying the molecular mechanisms of plasticity in striated muscles. Important questions in modern muscle biology are why some skeletal muscles atrophy and other muscles such as the diaphragm and the heart hypertrophy during hibernation, and why, despite the atrophy, skeletal muscle strength and fatigue resistance are maintained after arousal. The molecular mechanisms responsible are of interest both from a fundamental biological viewpoint and as a potential therapy for patients in critical care units. In this issue of Experimental Physiology, Yan et al. (2021) shed light on the distinctive features of the molecular mechanisms involved in the development of the hypertrophic response in the diaphragm and the atrophic response in the gastrocnemius muscle in hibernating Daurian ground squirrels. In particular, the authors showed that a decrease in protein synthesis, an increase in protein degradation and a decrease in muscle regeneration potential contributed to the atrophy of the gastrocnemius muscles during torpor, while an increase in protein synthesis, a decrease in protein degradation and an increase in muscle regeneration potential contributed to diaphragm muscle hypertrophy. In discussing these new and interesting results, it can be assumed, however, that a significant contribution to the formation of hypertrophic or atrophic muscle phenotype could have been made by changes in anabolic and catabolic processes occurring in interbout arousals, as indicated by the results of recent studies (Chang et al., 2020; Popova et al., 2020 ). Further research is needed to show how important interbout arousals are in the regulation of protein turnover and regeneration in different striated muscles in hibernating animals. Understanding these and other molecular mechanisms is important for choosing strategies and methods for treatment of skeletal muscle pathologies and diseases of the cardiovascular system, as well as for avoiding injurious interventions in cases of energy deficits in critical illness (Stanzani et al., 2020). None declared.