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17 pages, 10557 KiB  
Article
Formation of an Amyloid-like Structure During In Vitro Interaction of Titin and Myosin-Binding Protein C
by Tatiana A. Uryupina, Liya G. Bobyleva, Nikita V. Penkov, Maria A. Timchenko, Azat G. Gabdulkhakov, Anna V. Glyakina, Vadim V. Rogachevsky, Alexey K. Surin, Oxana V. Galzitskaya, Ivan M. Vikhlyantsev and Alexander G. Bobylev
Int. J. Mol. Sci. 2025, 26(14), 6910; https://doi.org/10.3390/ijms26146910 - 18 Jul 2025
Viewed by 241
Abstract
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 [...] Read more.
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. Full article
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23 pages, 3515 KiB  
Article
Characterization of Biocalcium Microparticles from Saltwater Crocodile (Crocodylus porosus) Bone and Their Potential for Enhancing Fish Bologna Quality
by Theeraphol Senphan, Natthapong Mungmueang, Supatra Karnjanapratum, Sutee Wangtueai, Akkasit Jongjareonrak and Suthasinee Yarnpakdee
Foods 2025, 14(10), 1732; https://doi.org/10.3390/foods14101732 - 13 May 2025
Viewed by 799
Abstract
Saltwater crocodile (SC; Crocodylus porosus) bone, an underutilized by-product, can be converted into high-value bio-calcium (Biocal), serving as a potential source of calcium and minerals. This study aimed to produce SC bone Biocal as functional gel enhancer for fish bologna development and [...] Read more.
Saltwater crocodile (SC; Crocodylus porosus) bone, an underutilized by-product, can be converted into high-value bio-calcium (Biocal), serving as a potential source of calcium and minerals. This study aimed to produce SC bone Biocal as functional gel enhancer for fish bologna development and to increase calcium intake. The resulting bone powder was evaluated for physicochemical, microbiological, and molecular properties. Additionally, the textural, physicochemical, structural, and sensorial properties of the formulated fish bologna incorporating Biocal at varying levels (0–10% w/w) were also evaluated. Biocal, obtained as a fine white powder, had a 16.83% yield. Mineral analysis showed 26.25% calcium and 13.72% phosphorus, with no harmful metals or pathogens detected. X-ray diffraction confirmed hydroxyapatite with 69.92% crystallinity, while calcium bioavailability was measured at 22.30%. Amino acid analysis indicated high levels of glycine, proline, and hydroxyproline, essential for collagen support. The findings confirmed that SC bone Biocal is beneficial and safe for food fortification. Incorporating SC Biocal (2–10% w/w) significantly affected the fish bologna characteristics (p < 0.05). As the Biocal level increased, the gel strength, hardness, and shear force also increased. The addition of 6% (w/w) Biocal significantly improved the textural property, without a detrimental effect on the sensory attributes of the bologna gel (p < 0.05). SDS-PAGE analysis showed TGase-enhanced myosin heavy chain (MHC) cross-linking, particularly in combination with Biocal. Moreover, the enriched Biocal–bologna gel exhibited a finer and denser microstructure. Thus, SC Biocal, particularly at 6% (w/w), can serve as a functional gel enhancer in surimi-based products, without compromising organoleptic quality. Full article
(This article belongs to the Section Food Quality and Safety)
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15 pages, 2693 KiB  
Article
Mechanisms of Chimeric Cell Therapy in Duchenne Muscular Dystrophy
by Maria Siemionow, Anna Ziemiecka, Katarzyna Bożyk and Krzysztof Siemionow
Biomedicines 2024, 12(9), 1996; https://doi.org/10.3390/biomedicines12091996 - 2 Sep 2024
Viewed by 2871
Abstract
Despite scientific efforts, there is no cure for Duchenne muscular dystrophy (DMD), a lethal, progressive, X-linked genetic disorder caused by mutations in the dystrophin gene. DMD leads to cardiac and skeletal muscle weakness, resulting in premature death due to cardio-pulmonary complications. We have [...] Read more.
Despite scientific efforts, there is no cure for Duchenne muscular dystrophy (DMD), a lethal, progressive, X-linked genetic disorder caused by mutations in the dystrophin gene. DMD leads to cardiac and skeletal muscle weakness, resulting in premature death due to cardio-pulmonary complications. We have developed Dystrophin Expressing Chimeric (DEC) cell therapy, DT-DEC01, by fusing human myoblasts from healthy donors and from DMD patients. Preclinical studies on human DEC cells showed increased dystrophin expression and improved cardiac, pulmonary, and skeletal muscle function after intraosseous administration. Our clinical study confirmed the safety and efficacy of DT-DEC01 therapy up to 24 months post-administration. In this study, we conducted in vitro assays to test the composition and potency of DT-DEC01, assessing chimerism level and the presence of dystrophin, desmin, and myosin heavy chain. Myoblast fusion resulted in the transfer of healthy donor mitochondria and the creation of chimeric mitochondria within DT-DEC01. The Pappenheim assay confirmed myotube formation in the final product. This study highlights the unique properties of DT-DEC01 therapy and their relevance to DMD treatment mechanisms. Full article
(This article belongs to the Special Issue Diagnosis, Pathogenesis and Treatment of Muscular Dystrophy)
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12 pages, 2056 KiB  
Communication
NLRX1 Mediates the Disruption of Intestinal Mucosal Function Caused by Porcine Astrovirus Infection via the Extracellular Regulated Protein Kinases/Myosin Light–Chain Kinase (ERK/MLCK) Pathway
by Jie Tao, Jinghua Cheng, Ying Shi, Benqiang Li, Pan Tang, Jiajie Jiao and Huili Liu
Cells 2024, 13(11), 913; https://doi.org/10.3390/cells13110913 - 25 May 2024
Viewed by 1879
Abstract
Porcine astrovirus (PAstV) has a potential zoonotic risk, with a high proportion of co-infection occurring with porcine epidemic diarrhea virus (PEDV) and other diarrheal pathogens. Despite its high prevalence, the cellular mechanism of PAstV pathogenesis is ill–defined. Previous proteomics [...] Read more.
Porcine astrovirus (PAstV) has a potential zoonotic risk, with a high proportion of co-infection occurring with porcine epidemic diarrhea virus (PEDV) and other diarrheal pathogens. Despite its high prevalence, the cellular mechanism of PAstV pathogenesis is ill–defined. Previous proteomics analyses have revealed that the differentially expressed protein NOD–like receptor X1 (NLRX1) located in the mitochondria participates in several important antiviral signaling pathways in PAstV–4 infection, which are closely related to mitophagy. In this study, we confirmed that PAstV–4 infection significantly up-regulated NLRX1 and mitophagy in Caco–2 cells, while the silencing of NLRX1 or the treatment of mitophagy inhibitor 3–MA inhibited PAstV–4 replication. Additionally, PAstV–4 infection triggered the activation of the extracellular regulated protein kinases/ myosin light-chain kinase (ERK/MLCK) pathway, followed by the down-regulation of tight–junction proteins (occludin and ZO–1) as well as MUC–2 expression. The silencing of NLRX1 or the treatment of 3–MA inhibited myosin light-chain (MLC) phosphorylation and up-regulated occludin and ZO–1 proteins. Treatment of the ERK inhibitor PD98059 also inhibited MLC phosphorylation, while MLCK inhibitor ML-7 mitigated the down-regulation of mucosa-related protein expression induced by PAstV–4 infection. Yet, adding PD98059 or ML–7 did not affect NLRX1 expression. In summary, this study preliminarily explains that NLRX1 plays an important role in the disruption of intestinal mucosal function triggered by PAstV–4 infection via the ERK/MLC pathway. It will be helpful for further antiviral drug target screening and disease therapy. Full article
(This article belongs to the Special Issue Charming Micro-Insights into Health and Diseases)
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21 pages, 1001 KiB  
Review
Emerging Concepts of Mechanisms Controlling Cardiac Tension: Focus on Familial Dilated Cardiomyopathy (DCM) and Sarcomere-Directed Therapies
by R. John Solaro, Paul H. Goldspink and Beata M. Wolska
Biomedicines 2024, 12(5), 999; https://doi.org/10.3390/biomedicines12050999 - 2 May 2024
Cited by 5 | Viewed by 2601
Abstract
Novel therapies for the treatment of familial dilated cardiomyopathy (DCM) are lacking. Shaping research directions to clinical needs is critical. Triggers for the progression of the disorder commonly occur due to specific gene variants that affect the production of sarcomeric/cytoskeletal proteins. Generally, these [...] Read more.
Novel therapies for the treatment of familial dilated cardiomyopathy (DCM) are lacking. Shaping research directions to clinical needs is critical. Triggers for the progression of the disorder commonly occur due to specific gene variants that affect the production of sarcomeric/cytoskeletal proteins. Generally, these variants cause a decrease in tension by the myofilaments, resulting in signaling abnormalities within the micro-environment, which over time result in structural and functional maladaptations, leading to heart failure (HF). Current concepts support the hypothesis that the mutant sarcomere proteins induce a causal depression in the tension-time integral (TTI) of linear preparations of cardiac muscle. However, molecular mechanisms underlying tension generation particularly concerning mutant proteins and their impact on sarcomere molecular signaling are currently controversial. Thus, there is a need for clarification as to how mutant proteins affect sarcomere molecular signaling in the etiology and progression of DCM. A main topic in this controversy is the control of the number of tension-generating myosin heads reacting with the thin filament. One line of investigation proposes that this number is determined by changes in the ratio of myosin heads in a sequestered super-relaxed state (SRX) or in a disordered relaxed state (DRX) poised for force generation upon the Ca2+ activation of the thin filament. Contrasting evidence from nanometer–micrometer-scale X-ray diffraction in intact trabeculae indicates that the SRX/DRX states may have a lesser role. Instead, the proposal is that myosin heads are in a basal OFF state in relaxation then transfer to an ON state through a mechano-sensing mechanism induced during early thin filament activation and increasing thick filament strain. Recent evidence about the modulation of these mechanisms by protein phosphorylation has also introduced a need for reconsidering the control of tension. We discuss these mechanisms that lead to different ideas related to how tension is disturbed by levels of mutant sarcomere proteins linked to the expression of gene variants in the complex landscape of DCM. Resolving the various mechanisms and incorporating them into a unified concept is crucial for gaining a comprehensive understanding of DCM. This deeper understanding is not only important for diagnosis and treatment strategies with small molecules, but also for understanding the reciprocal signaling processes that occur between cardiac myocytes and their micro-environment. By unraveling these complexities, we can pave the way for improved therapeutic interventions for managing DCM. Full article
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19 pages, 23274 KiB  
Article
Phosphorylated CPI-17 and MLC2 as Biomarkers of Coronary Artery Spasm–Induced Sudden Cardiac Death
by Yiming Dong, Jianfeng Wang, Chenteng Yang, Junxia Bao, Xia Liu, Hao Chen, Xiaojing Zhang, Weibo Shi, Lihua Zhang, Qian Qi, Yingmin Li, Songjun Wang, Rufei Ma, Bin Cong and Guozhong Zhang
Int. J. Mol. Sci. 2024, 25(5), 2941; https://doi.org/10.3390/ijms25052941 - 3 Mar 2024
Viewed by 1928
Abstract
Coronary artery spasm (CAS) plays an important role in the pathogeneses of various ischemic heart diseases and has gradually become a common cause of life-threatening arrhythmia. The specific molecular mechanism of CAS has not been fully elucidated, nor are there any specific diagnostic [...] Read more.
Coronary artery spasm (CAS) plays an important role in the pathogeneses of various ischemic heart diseases and has gradually become a common cause of life-threatening arrhythmia. The specific molecular mechanism of CAS has not been fully elucidated, nor are there any specific diagnostic markers for the condition. Therefore, this study aimed to examine the specific molecular mechanism underlying CAS, and screen for potential diagnostic markers. To this end, we successfully constructed a rat CAS model and achieved in vitro culture of a human coronary–artery smooth-muscle cell (hCASMC) contraction model. Possible molecular mechanisms by which protein kinase C (PKC) regulated CAS through the C kinase-potentiated protein phosphatase 1 inhibitor of 17 kDa (CPI-17)/myosin II regulatory light chain (MLC2) pathway were studied in vivo and in vitro to screen for potential molecular markers of CAS. We performed hematoxylin and eosin staining, myocardial zymogram, and transmission electron microscopy to determine myocardial and coronary artery injury in CAS rats. Then, using immunohistochemical staining, immunofluorescence staining, and Western blotting, we further demonstrated a potential molecular mechanism by which PKC regulated CAS via the CPI-17/MLC2 pathway. The results showed that membrane translocation of PKCα occurred in the coronary arteries of CAS rats. CPI-17/MLC2 signaling was observably activated in coronary arteries undergoing CAS. In addition, in vitro treatment of hCASMCs with angiotensin II (Ang II) increased PKCα membrane translocation while consistently activating CPI-17/MLC2 signaling. Conversely, GF-109203X and calphostin C, specific inhibitors of PKC, inactivated CPI-17/MLC2 signaling. We also collected the coronary artery tissues from deceased subjects suspected to have died of CAS and measured their levels of phosphorylated CPI-17 (p–CPI-17) and MLC2 (p-MLC2). Immunohistochemical staining was positive for p–CPI-17 and p-MLC2 in the tissues of these subjects. These findings suggest that PKCα induced CAS through the CPI-17/MLC2 pathway; therefore, p–CPI-17 and p-MLC2 could be used as potential markers for CAS. Our data provide novel evidence that therapeutic strategies against PKC or CPI-17/MLC2 signaling might be promising in the treatment of CAS. Full article
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15 pages, 610 KiB  
Article
Genetic Factors Associated with the Development of Neuropathy in Type 2 Diabetes
by Dóra Zsuszanna Tordai, Noémi Hajdú, Ramóna Rácz, Ildikó Istenes, Magdolna Békeffy, Orsolya Erzsébet Vági, Miklós Kempler, Anna Erzsébet Körei, Bálint Tóbiás, Anett Illés, Henriett Pikó, János Pál Kósa, Kristóf Árvai, Márton Papp, Péter András Lakatos, Péter Kempler and Zsuzsanna Putz
Int. J. Mol. Sci. 2024, 25(3), 1815; https://doi.org/10.3390/ijms25031815 - 2 Feb 2024
Cited by 7 | Viewed by 2944
Abstract
Neuropathy is a serious and frequent complication of type 2 diabetes (T2DM). This study was carried out to search for genetic factors associated with the development of diabetic neuropathy by whole exome sequencing. For this study, 24 patients with long-term type 2 diabetes [...] Read more.
Neuropathy is a serious and frequent complication of type 2 diabetes (T2DM). This study was carried out to search for genetic factors associated with the development of diabetic neuropathy by whole exome sequencing. For this study, 24 patients with long-term type 2 diabetes with neuropathy and 24 without underwent detailed neurological assessment and whole exome sequencing. Cardiovascular autonomic function was evaluated by cardiovascular reflex tests. Heart rate variability was measured by the triangle index. Sensory nerve function was estimated by Neurometer and Medoc devices. Neuropathic symptoms were characterized by the neuropathy total symptom score (NTSS). Whole exome sequencing (WES) was performed on a Thermo Ion GeneStudio S5 system determining the coding sequences of approximately 32,000 genes comprising 50 million base pairs. Variants were detected by Ion Reporter software and annotated using ANNOVAR, integrating database information from dbSNP, ClinVar, gnomAD, and OMIM. Integrative genomics viewer (IGV) was used for visualization of the mapped reads. We have identified genetic variants that were significantly associated with increased (22–49-fold) risk of neuropathy (rs2032930 and rs2032931 of recQ-mediated genome instability protein 2 (RMI2) gene), rs604349 of myosin binding protein H like (MYBPHL) gene and with reduced (0.07–0.08-fold) risk (rs917778 of multivesicular body subunit 12B (MVB12B) and rs2234753 of retinoic acid X receptor alpha (RXRA) genes). The rs2032930 showed a significant correlation with current perception thresholds measured at 5 Hz and 250 Hz for n. medianus (p = 0.042 and p = 0.003, respectively) and at 5 Hz for n. peroneus (p = 0.037), as well as the deep breath test (p = 0.022) and the NTSS (p = 0.023). The rs2032931 was associated with current perception thresholds (p = 0.003 and p = 0.037, respectively), deep breath test (p = 0.022), and NTSS (p = 0.023). The rs604349 correlated with values measured at 2000 (p = 0.049), 250 (p = 0.018), and 5 Hz (p = 0.005) for n. medianus, as well as warm perception threshold measured by Medoc device (p = 0.042). The rs2234753 showed correlations with a current perception threshold measured at 2000 Hz for n. medianus (p = 0.020), deep breath test (p = 0.040), and NTSS (p = 0.003). There was a significant relationship between rs91778 and cold perception threshold (p = 0.013). In our study, genetic variants have been identified that may have an impact on the risk of neuropathy developing in type 2 diabetic patients. These results could open up new opportunities for early preventive measures and might provide targets for new drug developments in the future. Full article
(This article belongs to the Section Molecular Genetics and Genomics)
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16 pages, 7207 KiB  
Communication
Efficacy of Rectal Systemic Administration of Mesenchymal Stem Cells to Injury Sites via the CXCL12/CXCR4 Axis to Promote Regeneration in a Rabbit Skeletal Muscle Injury Model
by Toru Ichiseki, Miyako Shimasaki, Shusuke Ueda, Hiroaki Hirata, Daisuke Souma, Norio Kawahara and Yoshimichi Ueda
Cells 2023, 12(13), 1729; https://doi.org/10.3390/cells12131729 - 27 Jun 2023
Cited by 3 | Viewed by 1871
Abstract
Mesenchymal stem cells (MSCs) have been transplanted directly into lesions or injected intravenously. The administration of MSCs using these delivery methods requires specialized knowledge, techniques, and facilities. Here, we describe intrarectal systemic administration of MSCs, a simple, non-invasive route for homing to the [...] Read more.
Mesenchymal stem cells (MSCs) have been transplanted directly into lesions or injected intravenously. The administration of MSCs using these delivery methods requires specialized knowledge, techniques, and facilities. Here, we describe intrarectal systemic administration of MSCs, a simple, non-invasive route for homing to the injury sites to promote the regeneration of skeletal muscle injuries. Using a cardiotoxin (CTX)-induced rabbit skeletal muscle injury model, homing to the site of muscle injury was confirmed by intrarectal administration of MSCs; the time required for homing after intrarectal administration was approximately 5 days. In addition, the C-X-C chemokine ligand 12 (CXCL12)/C-X-C chemokine receptor-4 (CXCR4) axis was found to be involved in the homing process. Histopathological examinations showed that skeletal muscle regeneration was promoted in the MSCs-administered group compared to the CTX-only group. Myosin heavy polypeptide 3 (Myh3) expression, an indicator of early muscle regeneration, was detected earlier in the intrarectal MSCs group compared to the CTX-only group. These findings indicate that intrarectal administration of MSCs is effective in homing to the injured area, where they promote injury repair. Since intrarectal administration is a simple and non-invasive delivery route, these findings may be valuable in future research on stem cell therapy. Full article
(This article belongs to the Collection Stem Cells in Tissue Engineering and Regeneration)
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22 pages, 5216 KiB  
Article
Curcumin Administration Improves Force of mdx Dystrophic Diaphragm by Acting on Fiber-Type Composition, Myosin Nitrotyrosination and SERCA1 Protein Levels
by Luisa Gorza, Elena Germinario, Maurizio Vitadello, Irene Guerra, Federica De Majo, Francesca Gasparella, Paolo Caliceti, Libero Vitiello and Daniela Danieli-Betto
Antioxidants 2023, 12(6), 1181; https://doi.org/10.3390/antiox12061181 - 30 May 2023
Cited by 5 | Viewed by 2172
Abstract
The vegetal polyphenol curcumin displays beneficial effects against skeletal muscle derangement induced by oxidative stress, disuse or aging. Since oxidative stress and inflammation are involved in the progression of muscle dystrophy, the effects of curcumin administration were investigated in the diaphragm of mdx [...] Read more.
The vegetal polyphenol curcumin displays beneficial effects against skeletal muscle derangement induced by oxidative stress, disuse or aging. Since oxidative stress and inflammation are involved in the progression of muscle dystrophy, the effects of curcumin administration were investigated in the diaphragm of mdx mice injected intraperitoneally or subcutaneously with curcumin for 4–12–24 weeks. Curcumin treatment independently of the way and duration of administration (i) ameliorated myofiber maturation index without affecting myofiber necrosis, inflammation and degree of fibrosis; (ii) counteracted the decrease in type 2X and 2B fiber percentage; (iii) increased about 30% both twitch and tetanic tensions of diaphragm strips; (iv) reduced myosin nitrotyrosination and tropomyosin oxidation; (v) acted on two opposite nNOS regulators by decreasing active AMP-Kinase and increasing SERCA1 protein levels, the latter effect being detectable also in myotube cultures from mdx satellite cells. Interestingly, increased contractility, decreased myosin nitrotyrosination and SERCA1 upregulation were also detectable in the mdx diaphragm after a 4-week administration of the NOS inhibitor 7-Nitroindazole, and were not improved further by a combined treatment. In conclusion, curcumin has beneficial effects on the dystrophic muscle, mechanistically acting for the containment of a deregulated nNOS activity. Full article
(This article belongs to the Special Issue Regulatory Effects of Curcumin)
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19 pages, 4547 KiB  
Article
Using Multiscale Simulations as a Tool to Interpret Equatorial X-ray Fiber Diffraction Patterns from Skeletal Muscle
by Momcilo Prodanovic, Yiwei Wang, Srboljub M. Mijailovich and Thomas Irving
Int. J. Mol. Sci. 2023, 24(10), 8474; https://doi.org/10.3390/ijms24108474 - 9 May 2023
Cited by 4 | Viewed by 2163
Abstract
Synchrotron small-angle X-ray diffraction is the method of choice for nm-scale structural studies of striated muscle under physiological conditions and on millisecond time scales. The lack of generally applicable computational tools for modeling X-ray diffraction patterns from intact muscles has been a significant [...] Read more.
Synchrotron small-angle X-ray diffraction is the method of choice for nm-scale structural studies of striated muscle under physiological conditions and on millisecond time scales. The lack of generally applicable computational tools for modeling X-ray diffraction patterns from intact muscles has been a significant barrier to exploiting the full potential of this technique. Here, we report a novel “forward problem” approach using the spatially explicit computational simulation platform MUSICO to predict equatorial small-angle X-ray diffraction patterns and the force output simultaneously from resting and isometrically contracting rat skeletal muscle that can be compared to experimental data. The simulation generates families of thick–thin filament repeating units, each with their individually predicted occupancies of different populations of active and inactive myosin heads that can be used to generate 2D-projected electron density models based on known Protein Data Bank structures. We show how, by adjusting only a few selected parameters, we can achieve a good correspondence between experimental and predicted X-ray intensities. The developments presented here demonstrate the feasibility of combining X-ray diffraction and spatially explicit modeling to form a powerful hypothesis-generating tool that can be used to motivate experiments that can reveal emergent properties of muscle. Full article
(This article belongs to the Special Issue Muscle Proteins, Functions and Interactions)
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25 pages, 11112 KiB  
Article
Unleashed Actin Assembly in Capping Protein-Deficient B16-F1 Cells Enables Identification of Multiple Factors Contributing to Filopodium Formation
by Jens Ingo Hein, Jonas Scholz, Sarah Körber, Thomas Kaufmann and Jan Faix
Cells 2023, 12(6), 890; https://doi.org/10.3390/cells12060890 - 14 Mar 2023
Cited by 5 | Viewed by 3657
Abstract
Background: Filopodia are dynamic, finger-like actin-filament bundles that overcome membrane tension by forces generated through actin polymerization at their tips to allow extension of these structures a few microns beyond the cell periphery. Actin assembly of these protrusions is regulated by accessory proteins [...] Read more.
Background: Filopodia are dynamic, finger-like actin-filament bundles that overcome membrane tension by forces generated through actin polymerization at their tips to allow extension of these structures a few microns beyond the cell periphery. Actin assembly of these protrusions is regulated by accessory proteins including heterodimeric capping protein (CP) or Ena/VASP actin polymerases to either terminate or promote filament growth. Accordingly, the depletion of CP in B16-F1 melanoma cells was previously shown to cause an explosive formation of filopodia. In Ena/VASP-deficient cells, CP depletion appeared to result in ruffling instead of inducing filopodia, implying that Ena/VASP proteins are absolutely essential for filopodia formation. However, this hypothesis was not yet experimentally confirmed. Methods: Here, we used B16-F1 cells and CRISPR/Cas9 technology to eliminate CP either alone or in combination with Ena/VASP or other factors residing at filopodia tips, followed by quantifications of filopodia length and number. Results: Unexpectedly, we find massive formations of filopodia even in the absence of CP and Ena/VASP proteins. Notably, combined inactivation of Ena/VASP, unconventional myosin-X and the formin FMNL3 was required to markedly impair filopodia formation in CP-deficient cells. Conclusions: Taken together, our results reveal that, besides Ena/VASP proteins, numerous other factors contribute to filopodia formation. Full article
(This article belongs to the Special Issue Cellular Integrity under Mechanical Stress)
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20 pages, 8670 KiB  
Article
The Flavonoid Naringenin Alleviates Collagen-Induced Arthritis through Curbing the Migration and Polarization of CD4+ T Lymphocyte Driven by Regulating Mitochondrial Fission
by Yue-Peng Jiang, Jun-Jun Wen, Xiao-Xuan Zhao, Yuan-Cheng Gao, Xiao Ma, Si-Yue Song, Yan Jin, Tie-Juan Shao, Jie Yu and Cheng-Ping Wen
Int. J. Mol. Sci. 2023, 24(1), 279; https://doi.org/10.3390/ijms24010279 - 23 Dec 2022
Cited by 20 | Viewed by 3454
Abstract
Rheumatoid arthritis (RA) is a progressive autoimmune disease. Due to local infiltration and damage to the joints, activated CD4+ T cells play a crucial role in the progression of RA. However, the exact regulatory mechanisms are perplexing, which makes the effective management [...] Read more.
Rheumatoid arthritis (RA) is a progressive autoimmune disease. Due to local infiltration and damage to the joints, activated CD4+ T cells play a crucial role in the progression of RA. However, the exact regulatory mechanisms are perplexing, which makes the effective management of RA frustrating. This study aimed to investigate the effect of mitochondria fission on the polarization and migration of CD4+ T cells as well as the regulatory mechanism of NAR, so as to provide enlightenment on therapeutic targets and novel strategies for the treatment of RA. In this study, a collagen-induced arthritis (CIA) model was established, and rats were randomly given saline or naringenin (NAR, 10 mg/kg, 20 mg/kg, 50 mg/kg, i.p.) once a day, before being euthanized on the 42nd day of primary immunization. The pain-like behavior, articular index scores, account of synovial-infiltrated CD4+ T cells, and inflammatory factors were investigated in each group. In vitro, spleen CD4+ T lymphocytes were derived from each group. In addition, mitochondrial division inhibitor 1 (Mdivi-1) or NAR was added to the cell medium containing C-X-C motif chemokine ligand 12 (CXCL12) in order to induce CD4+ T lymphocytes, respectively. The polarization capacity of CD4+ T cells was evaluated through the immunofluorescence intensity of the F-actin and myosin light chain phosphorylated at Ser19 (pMLC S19), and the mitochondrial distribution was determined by co-localization analysis of the translocase of outer mitochondrial membrane 20 (TOM20, the mitochondrial marker) and intercellular adhesion molecule 1 (ICAM1, the uropod marker). The mitochondrial fission was investigated by detecting dynamin-related protein 1 (Drp1) and mitochondrial fission protein 1 (Fis1) using Western blot and immunofluorescence. This study revealed that high-dose NAR (50 mg/kg, i.p.) alleviated pain-like behavior and articular index scores, reduced the serum level of interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α), and accounted for CD4+ T lymphocytes that infiltrated into the synovial membrane of the CIA group. Meanwhile, NAR (50 mg/kg, i.p.) suppressed the polarization of spleen CD4+ T lymphocytes, reduced the redistribution of mitochondria in the uropod, and inhibited the expression of Drp1 and Fis1 in the CIA model. Furthermore, the in vitro experiments confirmed that NAR reduced mitochondrial fission, which in turn inhibited the CXCL12-induced polarization and migration of CD4+ T lymphocytes. Our results demonstrated that the flavonoid NAR was a promising drug for the treatment of RA, which could effectively interfere with mitochondrial fission, thus inhibiting the polarization and migration of CD4+ T cells in the synovial membrane. Full article
(This article belongs to the Section Biochemistry)
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14 pages, 2990 KiB  
Article
MgADP Promotes Myosin Head Movement toward Actin at Low [Ca2+] to Increase Force Production and Ca2+-Sensitivity of Contraction in Permeabilized Porcine Myocardial Strips
by Peter O. Awinda, Weikang Ma, Kyrah L. Turner, Jing Zhao, Henry Gong, Mindy S. Thompson, Kenneth S. Campbell, Thomas C. Irving and Bertrand C. W. Tanner
Int. J. Mol. Sci. 2022, 23(23), 15084; https://doi.org/10.3390/ijms232315084 - 1 Dec 2022
Cited by 3 | Viewed by 2250
Abstract
Myosin cross-bridges dissociate from actin following Mg2+-adenosine triphosphate (MgATP) binding. Myosin hydrolyses MgATP into inorganic phosphate (Pi) and Mg2+-adenosine diphosphate (ADP), and release of these hydrolysis products drives chemo-mechanical energy transitions within the cross-bridge cycle to power [...] Read more.
Myosin cross-bridges dissociate from actin following Mg2+-adenosine triphosphate (MgATP) binding. Myosin hydrolyses MgATP into inorganic phosphate (Pi) and Mg2+-adenosine diphosphate (ADP), and release of these hydrolysis products drives chemo-mechanical energy transitions within the cross-bridge cycle to power muscle contraction. Some forms of heart disease are associated with metabolic or enzymatic dysregulation of the MgATP-MgADP nucleotide pool, resulting in elevated cytosolic [MgADP] and impaired muscle relaxation. We investigated the mechanical and structural effects of increasing [MgADP] in permeabilized myocardial strips from porcine left ventricle samples. Sarcomere length was set to 2.0 µm at 28 °C, and all solutions contained 3% dextran T-500 to compress myofilament lattice spacing to near-physiological values. Under relaxing low [Ca2+] conditions (pCa 8.0, where pCa = −log10[Ca2+]), tension increased as [MgADP] increased from 0-5 mM. Complementary small-angle X-ray diffraction measurements show that the equatorial intensity ratio, I1,1/I1,0, also increased as [MgADP] increased from 0 to 5 mM, indicating myosin head movement away from the thick-filament backbone towards the thin-filament. Ca2+-activated force-pCa measurements show that Ca2+-sensitivity of contraction increased with 5 mM MgADP, compared to 0 mM MgADP. These data show that MgADP augments tension at low [Ca2+] and Ca2+-sensitivity of contraction, suggesting that MgADP destabilizes the quasi-helically ordered myosin OFF state, thereby shifting the cross-bridge population towards the disordered myosin ON state. Together, these results indicate that MgADP enhances the probability of cross-bridge binding to actin due to enhancement of both thick and thin filament-based activation mechanisms. Full article
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12 pages, 1655 KiB  
Article
EMD-57033 Augments the Contractility in Porcine Myocardium by Promoting the Activation of Myosin in Thick Filaments
by Vivek Jani, Wenjing Qian, Shengyao Yuan, Thomas Irving and Weikang Ma
Int. J. Mol. Sci. 2022, 23(23), 14517; https://doi.org/10.3390/ijms232314517 - 22 Nov 2022
Cited by 8 | Viewed by 2190
Abstract
Sufficient cardiac contractility is necessary to ensure the sufficient cardiac output to provide an adequate end-organ perfusion. Inadequate cardiac output and the diminished perfusion of vital organs from depressed myocardium contractility is a hallmark end-stage of heart failure. There are no available therapeutics [...] Read more.
Sufficient cardiac contractility is necessary to ensure the sufficient cardiac output to provide an adequate end-organ perfusion. Inadequate cardiac output and the diminished perfusion of vital organs from depressed myocardium contractility is a hallmark end-stage of heart failure. There are no available therapeutics that directly target contractile proteins to improve the myocardium contractility and reduce mortality. The purpose of this study is to present a proof of concept to aid in the development of muscle activators (myotropes) for augmenting the contractility in clinical heart failure. Here we use a combination of cardiomyocyte mechanics, the biochemical quantification of the ATP turnover, and small angle X-ray diffraction on a permeabilized porcine myocardium to study the mechanisms of EMD-57033 (EMD) for activating myosin. We show that EMD increases the contractility in a porcine myocardium at submaximal and systolic calcium concentrations. Biochemical assays show that EMD decreases the proportion of myosin heads in the energy sparing super-relaxed (SRX) state under relaxing conditions, which are less likely to interact with actin during contraction. Structural assays show that EMD moves the myosin heads in relaxed muscles from a structurally ordered state close to the thick filament backbone, to a disordered state closer to the actin filament, while simultaneously inducing structural changes in the troponin complex on the actin filament. The dual effects of EMD on activating myosin heads and the troponin complex provides a proof of concept for the use of small molecule muscle activators for augmenting the contractility in heart failure. Full article
(This article belongs to the Special Issue Sarcomeric Proteins in Health and Disease: 3rd Edition)
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18 pages, 2699 KiB  
Review
Genes Whose Gain or Loss of Function Changes Type 1, 2A, 2X, or 2B Muscle Fibre Proportions in Mice—A Systematic Review
by Gabryela Kuhnen, Tiago Guedes Russomanno, Marta Murgia, Nicolas J. Pillon, Martin Schönfelder and Henning Wackerhage
Int. J. Mol. Sci. 2022, 23(21), 12933; https://doi.org/10.3390/ijms232112933 - 26 Oct 2022
Cited by 7 | Viewed by 6399
Abstract
Adult skeletal muscle fibres are classified as type 1, 2A, 2X, and 2B. These classifications are based on the expression of the dominant myosin heavy chain isoform. Muscle fibre-specific gene expression and proportions of muscle fibre types change during development and in response [...] Read more.
Adult skeletal muscle fibres are classified as type 1, 2A, 2X, and 2B. These classifications are based on the expression of the dominant myosin heavy chain isoform. Muscle fibre-specific gene expression and proportions of muscle fibre types change during development and in response to exercise, chronic electrical stimulation, or inactivity. To identify genes whose gain or loss-of-function alters type 1, 2A, 2X, or 2B muscle fibre proportions in mice, we conducted a systematic review of transgenic mouse studies. The systematic review was conducted in accordance with the 2009 PRISMA guidelines and the PICO framework. We identified 25 “muscle fibre genes” (Akirin1, Bdkrb2, Bdnf, Camk4, Ccnd3, Cpt1a, Epas1, Esrrg, Foxj3, Foxo1, Il15, Mapk12, Mstn, Myod1, Ncor1, Nfatc1, Nol3, Ppargc1a, Ppargc1b, Sirt1, Sirt3, Thra, Thrb, Trib3, and Vgll2) whose gain or loss-of-function significantly changes type 1, 2A, 2X or 2B muscle fibre proportions in mice. The fact that 15 of the 25 muscle fibre genes are transcriptional regulators suggests that muscle fibre-specific gene expression is primarily regulated transcriptionally. A reanalysis of existing datasets revealed that the expression of Ppargc1a and Vgll2 increases and Mstn decreases after exercise, respectively. This suggests that these genes help to regulate the muscle fibre adaptation to exercise. Finally, there are many known DNA sequence variants of muscle fibre genes. It seems likely that such DNA sequence variants contribute to the large variation of muscle fibre type proportions in the human population. Full article
(This article belongs to the Special Issue Novel Molecular Approaches to Skeletal Muscle Disease and Disuse 2.0)
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