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Angiomatoid fibrous histiocytoma (AFH) is a soft tissue tumor of uncertain differentiation. Although its prognosis is good, its diagnosis and differential diagnosis remain a challenge, particularly for tumors with an atypical morphology. We evaluated the clinicopathological characteristics of 14 AFH cases and examined the key factors in its diagnosis or differential diagnosis. The cohort comprised 6 men and 8 women aged 9-65 years (average age: 31.2 years). Most of the tumors (11/14, 79%) were located in soft tissues, whereas 3/14 (21%) were located in the lung (1 case) and brain (2 cases). Tumor cells were spindle-shaped to epithelioid, with a visible fibrous capsule (9/14, 64%), hemorrhagic gap (9/14, 64%), lymphocyte sleeve (7/14, 50%), necrosis (3/14, 21%), and infiltrative boundary (4/14, 29%). The tumors expressed desmin (10/14, 71%) and exhibited low levels of Ki-67. 13 cases (93%) displayed ESWSR1 gene rearrangement. At follow-up, 1 case (7%) experienced local tumor recurrence. AFH is a rare intermediate tumor. Its pathological diagnosis requires a comprehensive analysis of histological, immunophenotypic, and molecular genetic features to avoid misdiagnosis. Our study has further enriched the histological features of AFH, emphasizing the importance of differential diagnosis and providing a reference for clinical practice., (© 2024. The Author(s) under exclusive licence to The Japanese Society for Clinical Molecular Morphology.)

The purpose of this study was to establish a system for the isolation, culture, and differentiation of sheep myoblasts, and to explore the expression patterns as well as mutual relationships of muscle-specific genes. Sheep fetal myoblasts (SFMs) were isolated by two-step enzymatic digestion, purified by differential adhesion and identified using immunofluorescence techniques. Two percent horse serum was used to induce differentiation in SFMs. Real-time quantitative and Western blot analyses were respectively used to detect the mRNA and protein expressions of muscle-specific genes including MyoD, MyoG, Myf5, Myf6, PAX3, PAX7, myomaker, desmin, MYH1, MYH2, MYH4, MYH7, and MSTN during the differentiation of SFMs. Finally, the correlation between muscle-specific genes was analyzed by the Pearson correlation coefficient method. The results showed that the isolated and purified SFMs could form myotubes after the induction for differentiation. The marker factors including MyoD, MyoG, myomaker, desmin, and MyHC were positively stained in SFMs. The mRNA expressions of MyoD, MyoG, and myomaker increased and then decreased, while Myf5, PAX3, and PAX7 decreased; Myf6, desmin, MYH1, MYH2, MYH4, and MYH7 increased; and MSTN fluctuated up and down during the differentiation of SFMs. The expression patterns of protein were basically consistent with those of mRNA except MSTN. There existed significant or highly significant correlations at mRNA or protein level among some genes. Some transcription factor proteins (MyoD, Myf5, Myf6, PAX3, PAX7) showed significant or highly significant correlations with the mRNA level of some other genes and/or themselves. In conclusion, SFMs with good myogenic differentiation ability were successfully isolated, and the expression patterns and correlations of muscle-specific genes during SFM differentiation were revealed, which laid an important foundation for elucidating the gene regulation mechanism of sheep myogenesis., (© 2022. The Society for In Vitro Biology.)

Atherosclerosis (AS) is a dynamic and multi-stage process that involves various cells types, such as vascular smooth muscle cells (VSMCs) and molecules such as microRNAs. In this study, we investigated how miR-338-3p works in the process of AS. To determine how miR-338-3p was expressed in AS, an AS rat model was established and primary rat VSMCs were cultured. Real-time polymerase chain reaction was performed to detect miR-338-3p expression. Markers of different VSMC phenotypes were tested by Western blot. Immunofluorescent staining was employed to observe the morphologic changes of VSMCs transfected with miR-338-3p mimics. A dual luciferase reporter assay system was used to verify that desmin was a target of miR-338-3p. To further identify the role of miR-338-3p in the development of AS, VSMC proliferation and migration were evaluated by EdU incorporation assay, MTT assay, and wound healing assay. miR-338-3p expression was upregulated in the aortic tissues of an AS rat model and in primary rat VSMCs from a later passage. The transfection of miR-338-3p mimics in VSMCs promoted the synthetic cell phenotype. Bioinformatics analysis proposed desmin as a candidate target for miR-338-3p and the dual luciferase reporter assay confirmed in vivo that desmin was a direct target of miR-338-3p. The MTT and EdU incorporation assay revealed increased cell viability when miR-338-3p mimics were transfected. The increased expression of PCNA was a consistent observation, although a positive result was not obtained with respect to VSMC mobility. In AS, miR-338-3p expression was elevated. Elevated miR-338-3p inhibited the expression of desmin, thus promoting the contractile-to-synthetic VSMC phenotypic transition. In addition to morphologic changes, miR-338-3p enhanced the proliferative but not mobile ability of VSMCs. In summary, miR-338-3p promotes the development of AS., (© 2021. The Author(s).)

Introduction and Hypothesis: An animal model of vaginal distention (VD) was developed to reproduce the acute urethral injury and deficiency underlying stress urinary incontinence (SUI). Data on the chronic effects of urethral trauma and the recovery process are still scarce. We investigated acute, short- and long-term histomorphological and molecular changes in the urethra of rats post 12-h intermittent VD., Methods: We evaluated the urethra of four groups of female rats (n = 72): control without trauma, 1 h, 7 days and 30 days post VD. We compared the gene and protein expression of the VEGF and NGF growth factors, collagens (COL1a1 and COL3a1), desmin, smooth muscle myosin (MYH11), skeletal muscle myosins (MYH1, MYH2 and MYH3) and cell proliferation marker MKi67. We used real-time RT-qPCR, and immunohistochemistry., Results: Histology showed urethral damage after VD mainly involving the muscular layers. VEGF, NGF, desmin and MKi67 mRNA were significantly upregulated in the urethras of rats 1-h post VD compared with controls (P < 0.05 for all). By 7 days post trauma, COL1a1, MYH11 and MYH3 genes were overexpressed compared with controls (p < 0.05 for all). The COL3a1 protein level was increased by 2.6 times by day 7, while MYH2 protein was significantly decreased (around two times) from 7 to 30 days post VD compared with controls (p < 0.05 for both)., Conclusions: The 12-h intermittent VD causes chronic alterations in the urethra represented by increased COL3a1 and decreased MYH2 protein levels in the long term. The model can potentially be used to study the mechanisms of urethral injury and recovery as well as the physiopathology of SUI.

We analyzed cultures of 5 independent myoblast lines from human skeletal muscles. It was shown that the content of desmin-positive cells in cultures at early passages exceeds 90%. Typical morphofunctional signs of myogenic differentiation disturbances were identified and their dynamics was studied. Signs of alternative adipogenic and chondrogenic differentiation of cells were revealed. Based on these data, limitations for the use of myoblast cultures of certain passages for biomedical research and cell therapy were evaluated.

Mutations in the small heat shock protein chaperone CRYAB (αB-crystallin/HSPB5) and the intermediate filament protein desmin, phenocopy each other causing cardiomyopathies. Whilst the binding sites for desmin on CRYAB have been determined, desmin epitopes responsible for CRYAB binding and also the parameters that determine CRYAB binding to desmin filaments are unknown. Using a combination of co-sedimentation centrifugation, viscometric assays and electron microscopy of negatively stained filaments to analyse the in vitro assembly of desmin filaments, we show that the binding of CRYAB to desmin is subject to its assembly status, to the subunit organization within filaments formed and to the integrity of the C-terminal tail domain of desmin. Our in vitro studies using a rapid assembly protocol, C-terminally truncated desmin and two disease-causing mutants (I451M and R454W) suggest that CRYAB is a sensor for the surface topology of the desmin filament. Our data also suggest that CRYAB performs an assembly chaperone role because the assembling filaments have different CRYAB-binding properties during the maturation process. We suggest that the capability of CRYAB to distinguish between filaments with different surface topologies due either to mutation (R454W) or assembly protocol is important to understanding the pathomechanism(s) of desmin-CRYAB myopathies.

The therapeutic potential of mesenchymal stem cell-conditioned medium (MSC-CM) has been reported with various types of disease models. Here, we examine the therapeutic effect of umbilical cord MSC-CM (UCMSC-CM) on muscle-related disease, using a dexamethasone (Dex)-induced muscle atrophy in vitro model. The expressions of muscle atrophy-related proteins (MuRF-1 and MAFbx) and muscle-specific proteins (desmin and myogenin) were evaluated by Western blot analysis. The level of production of reactive oxygen species (ROS) was determined using a 2',7'-dichlorofluorescein diacetate (DCFDA) dye assay. The expression of antioxidant enzymes (copper/zinc-superoxide dismutase (Cu/Zn-SOD), manganese superoxide dismutase (MnSOD), glutathione peroxidase-1 (GPx-1), and catalase (CAT)) was verified by reverse transcription polymerase chain reaction (RT-PCR). When L6 cells were exposed to Dex, the expression of muscle atrophy-related proteins was increased by 50-70%, and the expression of muscle-specific proteins was in turn decreased by 23-40%. Conversely, when the L6 cells were co-treated with UCMSC-CM and Dex, the expression of muscle atrophy-related proteins was reduced in a UCMSC-CM dose-dependent manner and the expression of muscle-specific proteins was restored to near-normal levels. Moreover, ROS generation was effectively suppressed and the expression of antioxidant enzymes was recovered to a normal degree. These data imply that UCMSC-CM clearly has the potential to prevent muscle atrophy. Thus, our present study offers fundamental data on the potential treatment of muscle-related disease using UCMSC-CM.

Desmin, the muscle-specific intermediate filament, is involved in myofibrillar myopathies, dilated cardiomyopathy and muscle wasting. Desmin is the target of posttranslational modifications (PTMs) such as phosphorylation, ADP-ribosylation and ubiquitylation as well as nonenzymatic modifications such as glycation, oxidation and nitration. Several PTM target residues and their corresponding modifying enzymes have been discovered in human and nonhuman desmin. The major effect of phosphorylation and ADP-ribosylation is the disassembly of desmin filaments, while ubiquitylation of desmin leads to its degradation. The regulation of the desmin filament network by phosphorylation and ADP-ribosylation was found to be implicated in several major biological processes such as myogenesis, myoblast fusion, muscle contraction, muscle atrophy, cell division and possibly desmin interactions with its binding partners. Phosphorylation of desmin is also implicated in many forms of desmin-related myopathies (desminopathies). In this review, we summarize the findings on desmin PTMs and their implication in biological processes and pathologies, and discuss the current knowledge on the regulation of the desmin network by PTMs. We conclude that the desmin filament network can be seen as an intricate scaffold for muscle cell structure and biological processes and that its dynamics can be affected by PTMs. There are now precise tools to investigate PTMs and visualize cellular structures that have been underexploited in the study of desminopathies. Future studies should focus on these aspects.

In search of early structural markers of arteriogenesis, we studied the expression of gap junction proteins as well as of contractile and cytoskeletal proteins in smooth muscle cells (SMCs) during coronary collateral vessel growth induced by chronic occlusion of the left circumflex artery (LCx) in the dog heart. We used confocal microscopy with antibodies against connexin37 (Cx37), alpha-smooth muscle actin (alpha-SM actin), calponin, desmin and vinculin. The quantitative confocal analysis of immunofluorescence intensity showed that (1) in normal vessels (NV), Cx37 was present in endothelium only, not in SMC. Calponin, alpha-SM actin, desmin and vinculin were evenly expressed in SMC. (2) In early growing V (EV) with minimal intima formation, alpha-SM actin, calponin and vinculin showed little change in SMC, but desmin was 3.3 times lower than in NV, and Cx37 was induced (NV 0 arbitrary units/microm2, EV 50.3). (3) In actively growing V (AV), alpha-SM actin, calponin and vinculin were 3-, 3.3- and 2.9-fold lower, respectively, in the neointima as compared to the media. However, Cx37 was 48.2 AU/microm2 in the media and 15.8 AU/microm2 in the neointima. Desmin was almost absent in the neointima and 5-fold reduced in the media. SMC, strongly positive for alpha-SM actin and calponin, expressed Cx37. Our findings indicate that induction of Cx37 and reduction of desmin precede the phenotypic changes of SMCs, which are characterized by down-regulation of alpha-SM actin, calponin and vinculin, and the formation of a neointima. An altered expression of Cx37 and desmin, therefore, are early markers for arteriogenesis in dog heart.

Passive stiffness was found to be increased in mouse soleus muscles lacking desmin. Because titin is considered to be the major source of muscle elasticity, the stiffening might be explainable by titin adaptation. To test this, passive mechanical properties of single skinned fibres of soleus muscles from desmin knockout and control mice were analysed by using various extension tests. Titin expression was studied by SDS-gel electrophoresis. Absence of desmin did not modify either electrophoretic mobility of the titin band (3700 kDa) or optical density-unit ratios between bands for titin and nebulin (congruent with 0.3) and bands for titin and myosin heavy chain (congruent with 0.08). Elastic properties of fibres were not altered in the absence of desmin since passive tensions were similar under quasi-static (56-66 kN m(-2)) and dynamic (100-118 kN m(-2)) conditions whatever the kind of fibre. Thus, titin is unlikely to be responsible for the large increase in passive stiffness observed in whole soleus muscles when desmin is lacking.

In heart tissue from mice lacking the intermediate filament (IF) desmin, mitochondria show an abnormal shape and distribution (Thornell et al., 1997). In the present study we have isolated heart mitochondria from desmin null (D-/-) and control (D+/+) mice, and analyzed their composition by SDS-PAGE, immunoblotting, and enzyme measurements. We found both in vitro and in situ that the conventional kinesin, the microtubule-associated plus-end directed motor, was frequently associated with D+/+ heart mitochondria, but not with D-/- heart mitochondria, suggesting that the positioning of mitochondria in heart is a dynamic event involving the IF desmin, the molecular motor kinesin, and, most likely, the microtubules (MT) network. Furthermore, an increased capacity in energy production was found, as indicated by a threefold higher creatine kinase activity in heart mitochondria from D-/- compared to D+/+ mice. We also observed a significantly lower amount of cytochrome c in heart mitochondria from D-/- mice, and a relocalization of Bcl-2, which may indicate an apoptotic condition in the cell leading to the earlier reported pathological events, such as cardiomyocytes degeneration and calcinosis of the heart (Thornell et al., 1997).

Mice lacking the gene encoding for the intermediate filament protein desmin have a surprisingly normal myofibrillar organization in skeletal muscle fibers, although myopathy develops in highly used muscles. In the present study we examined how synemin, paranemin, and plectin, three key cytoskeletal proteins related to desmin, are organized in normal and desmin knock-out (K/O) mice. We show that in wild-type mice, synemin, paranemin, and plectin were colocalized with desmin in Z-disc-associated striations and at the sarcolemma. All three proteins were also present at the myotendinous junctions and in the postsynaptic area of motor endplates. In the desmin K/O mice the distribution of plectin was unaffected, whereas synemin and paranemin were partly affected. The Z-disc-associated striations were in general no longer present in between the myofibrils. In contrast, at the myotendinous and neuromuscular junctions synemin and paranemin were still present. Our study shows that plectin differs from synemin and paranemin in its binding properties to the myofibrillar Z-discs and that the cytoskeleton in junctional areas is particularly complex in its organization.

Intermediate filament (IF) proteins are encoded by a large multigene family and form polymers with a uniform diameter of approximately 10 nm. However, although the cytoplasmic representatives all confirm to a unit-type structural principle leading to the formation of extended coiled coils, it is becoming increasingly clear that subunit arrangements and physical properties vary among the different filaments. Thus, the intricate tissue-specific expression pattern of individual IF proteins (especially, their co-expression with other members of the IF protein family or with IF-associated proteins to form obligatory heteropolymers) points to distinct functions acquired during evolution relevant to cellular homeostasis in various tissues.

Mice carrying chimeric, truncated or mutated genes encoding intermediate filament (IF) proteins type III do not show any detectable severe pathology. However, upon (over)expression of the transgene in the eye lens all animals develop lens opacification (cataract). At the cellular level the loss of visual acuity is preceded by interference with the terminal differentiation of lens fibre cells, plasma membrane damage, distorted assembly of the IF cytoskeleton and perturbation of the cytoskeleton-membrane complex. The degree of expression is paralleled by the extent of the damages.