scholarly journals Generating intrafusal skeletal muscle fibres in vitro: Current state of the art and future challenges

2020 ◽  
Vol 11 ◽  
pp. 204173142098520
Author(s):  
Philip Barrett ◽  
Tom J Quick ◽  
Vivek Mudera ◽  
Darren J Player
Keyword(s):  
Skeletal Muscle ◽  
Muscle Spindle ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Ex Vivo ◽  
Muscle Fibres ◽  
Current State ◽  
Intrafusal Fibres ◽  
Proprioceptive Function

Intrafusal fibres are a specialised cell population in skeletal muscle, found within the muscle spindle. These fibres have a mechano-sensory capacity, forming part of the monosynaptic stretch-reflex arc, a key component responsible for proprioceptive function. Impairment of proprioception and associated dysfunction of the muscle spindle is linked with many neuromuscular diseases. Research to-date has largely been undertaken in vivo or using ex vivo preparations. These studies have provided a foundation for our understanding of muscle spindle physiology, however, the cellular and molecular mechanisms which underpin physiological changes are yet to be fully elucidated. Therefrom, the use of in vitro models has been proposed, whereby intrafusal fibres can be generated de novo. Although there has been progress, it is predominantly a developing and evolving area of research. This narrative review presents the current state of art in this area and proposes the direction of future work, with the aim of providing novel pre-clinical and clinical applications.

scholarly journals Neuregulin 1 Drives Morphological and Phenotypical Changes in C2C12 Myotubes: Towards De Novo Formation of Intrafusal Fibres In Vitro

2022 ◽  
Vol 9 ◽  
Author(s):  
Philip Barrett ◽  
Tom J. Quick ◽  
Vivek Mudera ◽  
Darren J. Player
Keyword(s):  
Skeletal Muscle ◽  
Muscle Spindle ◽  
De Novo ◽  
C2c12 Myotubes ◽  
Muscle Fibres ◽  
Neuregulin 1 ◽  
Gene And Protein Expression ◽  
Intrafusal Fibres

Muscle spindles are sensory organs that detect and mediate both static and dynamic muscle stretch and monitor muscle position, through a specialised cell population, termed intrafusal fibres. It is these fibres that provide a key contribution to proprioception and muscle spindle dysfunction is associated with multiple neuromuscular diseases, aging and nerve injuries. To date, there are few publications focussed on de novo generation and characterisation of intrafusal muscle fibres in vitro. To this end, current models of skeletal muscle focus on extrafusal fibres and lack an appreciation for the afferent functions of the muscle spindle. The goal of this study was to produce and define intrafusal bag and chain myotubes from differentiated C2C12 myoblasts, utilising the addition of the developmentally associated protein, Neuregulin 1 (Nrg-1). Intrafusal bag myotubes have a fusiform shape and were assigned using statistical morphological parameters. The model was further validated using immunofluorescent microscopy and western blot analysis, directed against an extensive list of putative intrafusal specific markers, as identified in vivo. The addition of Nrg-1 treatment resulted in a 5-fold increase in intrafusal bag myotubes (as assessed by morphology) and increased protein and gene expression of the intrafusal specific transcription factor, Egr3. Surprisingly, Nrg-1 treated myotubes had significantly reduced gene and protein expression of many intrafusal specific markers and showed no specificity towards intrafusal bag morphology. Another novel finding highlights a proliferative effect for Nrg-1 during the serum starvation-initiated differentiation phase, leading to increased nuclei counts, paired with less myotube area per myonuclei. Therefore, despite no clear collective evidence for specific intrafusal development, Nrg-1 treated myotubes share two inherent characteristics of intrafusal fibres, which contain increased satellite cell numbers and smaller myonuclear domains compared with their extrafusal neighbours. This research represents a minimalistic, monocellular C2C12 model for progression towards de novo intrafusal skeletal muscle generation, with the most extensive characterisation to date. Integration of intrafusal myotubes, characteristic of native, in vivo intrafusal skeletal muscle into future biomimetic tissue engineered models could provide platforms for developmental or disease state studies, pre-clinical screening, or clinical applications.

scholarly journals Influence of Intermittent Cold Stimulations on CREB and Its Targeting Genes in Muscle: Investigations into Molecular Mechanisms of Local Cryotherapy

2019 ◽  
Author(s):  
Takehito Sugasawa ◽  
Tome Yoshiya ◽  
Yoshinori Takeuchi ◽  
Naoya Yahagi ◽  
Rahul Sharma ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Binding Protein ◽  
Cold Stimulation ◽  
Vitro Assay ◽  
Mitochondrial Dna Copy Number ◽  
Local Cryotherapy

Local cryotherapy is widely used as a treatment for sports-related skeletal muscle injury. However, its molecular mechanisms are unknown. To clarify these mechanisms, in this study, we applied one to three 15-min cold stimulations at 4 °C to various cell lines (in vitro), the tibialis anterior (TA) muscle (ex vivo), and mouse limbs (in vivo). In the in vitro assay, cAMP response element-binding protein 1 (CREB1) was markedly phosphorylated (as pCREB1) and CREB-binding protein (CBP) was recruited to pCREB-1 in response to two or three cold stimulations. In a reporter assay with the cAMP-responsive element, the signals significantly increased after two to three cold stimulations at 4 °C. In the ex vivo study, CREB-targeting genes were significantly upregulated following two or three cold stimulations. The in vivo experiment disclosed that cold stimulation of a mouse limb for 9 days significantly increased mitochondrial DNA copy number and upregulated genes such as Pgc-1α involved in mitochondrial biogenesis. The foregoing results suggest that local cryotherapy increases CREB transcription and upregulates CREB-targeting genes in a manner dependent on cold stimulation frequency and duration. This information may serve as an impetus for further investigations into local cryotherapy as a treatment for sports-related skeletal muscle trauma.

scholarly journals Influence of Intermittent Cold Stimulations on CREB and Its Targeting Genes in Muscle: Investigations into Molecular Mechanisms of Local Cryotherapy

2020 ◽  
Vol 21 (13) ◽  
pp. 4588
Author(s):  
Takehito Sugasawa ◽  
Yoshiya Tome ◽  
Yoshinori Takeuchi ◽  
Yasuko Yoshida ◽  
Naoya Yahagi ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Binding Protein ◽  
Cold Stimulation ◽  
Vitro Assay ◽  
Mitochondrial Dna Copy Number ◽  
Local Cryotherapy

Local cryotherapy is widely used as a treatment for sports-related skeletal muscle injuries. The molecular mechanisms are unknown. To clarify these mechanisms, we applied one to three 15-min cold stimulations at 4 °C to various cell lines (in vitro), the tibialis anterior (TA) muscle (ex vivo), and mouse limbs (in vivo). In the in vitro assay, cyclic AMP (cAMP) response element binding protein 1 (CREB1) was markedly phosphorylated (p-CREB1), and the CREB-binding protein (CBP) was recruited to p-CREB-1 in response to two or three cold stimulations. In a reporter assay with the cAMP-responsive element, the signals significantly increased after two to three cold stimulations at 4 °C. In the ex vivo study, CREB-targeting genes were significantly upregulated following two or three cold stimulations. The in vivo experiment disclosed that cold stimulation of a mouse limb for 9 days significantly increased mitochondrial DNA copy number and upregulated genes involved in mitochondrial biogenesis. The results suggest that local cryotherapy increases CREB transcription and upregulates CREB-targeting genes, in a manner dependent on cold stimulation frequency and duration. This information will inform further investigations into local cryotherapy as a treatment for sports-related skeletal muscle trauma.

scholarly journals A Reduction in Selenoprotein S Amplifies the Inflammatory Profile of Fast-Twitch Skeletal Muscle in themdxDystrophic Mouse

2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Craig Robert Wright ◽  
Giselle Larissa Allsopp ◽  
Alex Bernard Addinsall ◽  
Natasha Lee McRae ◽  
Sofianos Andrikopoulos ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Transforming Growth Factor ◽  
Ex Vivo ◽  
Muscle Fibres ◽  
Muscle Fibre Size ◽  
Monocyte Chemoattractant Protein 1 ◽  
Selenoprotein S ◽  
Inflammatory Profile ◽  
Fast Twitch

Excessive inflammation is a hallmark of muscle myopathies, including Duchenne muscular dystrophy (DMD). There is interest in characterising novel genes that regulate inflammation due to their potential to modify disease progression. Gene polymorphisms inSelenoprotein S(Seps1) are associated with elevated proinflammatory cytokines, and in vitro SEPS1 is protective against inflammatory stress. Given that SEPS1 is highly expressed in skeletal muscle, we investigated whether the genetic reduction ofSeps1exacerbated inflammation in themdxmouse. F1 malemdxmice with a heterozygousSeps1deletion (mdx:Seps1−/+) were generated. Themdx:Seps1−/+mice had a 50% reduction in SEPS1 protein expression in hindlimb muscles. In the extensor digitorum longus (EDL) muscles, mRNA expression ofmonocyte chemoattractant protein 1(Mcp-1) (P=0.034), macrophage markerF4/80(P=0.030), andtransforming growth factor-β1(Tgf-β1) (P=0.056) were increased inmdx:Seps1−/+mice. This was associated with a reduction in muscle fibre size; however, ex vivo EDL muscle strength and endurance were unaltered. In dystrophic slow twitch soleus muscles, SEPS1 reduction had no effect on the inflammatory profile nor function. In conclusion, the genetic reduction ofSeps1appears to specifically exacerbate the inflammatory profile of fast-twitch muscle fibres, which are typically more vulnerable to degeneration in dystrophy.

scholarly journals Cell–cell coupling and DNA methylation abnormal phenotypes in the after-hours mice

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Federico Tinarelli ◽  
Elena Ivanova ◽  
Ilaria Colombi ◽  
Erica Barini ◽  
Edoardo Balzani ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Neuronal Networks ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Neuronal Cultures ◽  
Functional Impairments ◽  
After Hours ◽  
The Impact

Abstract Background DNA methylation has emerged as an important epigenetic regulator of brain processes, including circadian rhythms. However, how DNA methylation intervenes between environmental signals, such as light entrainment, and the transcriptional and translational molecular mechanisms of the cellular clock is currently unknown. Here, we studied the after-hours mice, which have a point mutation in the Fbxl3 gene and a lengthened circadian period. Methods In this study, we used a combination of in vivo, ex vivo and in vitro approaches. We measured retinal responses in Afh animals and we have run reduced representation bisulphite sequencing (RRBS), pyrosequencing and gene expression analysis in a variety of brain tissues ex vivo. In vitro, we used primary neuronal cultures combined to micro electrode array (MEA) technology and gene expression. Results We observed functional impairments in mutant neuronal networks, and a reduction in the retinal responses to light-dependent stimuli. We detected abnormalities in the expression of photoreceptive melanopsin (OPN4). Furthermore, we identified alterations in the DNA methylation pathways throughout the retinohypothalamic tract terminals and links between the transcription factor Rev-Erbα and Fbxl3. Conclusions The results of this study, primarily represent a contribution towards an understanding of electrophysiological and molecular phenotypic responses to external stimuli in the Afh model. Moreover, as DNA methylation has recently emerged as a new regulator of neuronal networks with important consequences for circadian behaviour, we discuss the impact of the Afh mutation on the epigenetic landscape of circadian biology.

scholarly journals Fishing for Targets of Alien Metabolites: A Novel Peroxisome Proliferator-Activated Receptor (PPAR) Agonist from a Marine Pest

Marine Drugs ◽  
2018 ◽  
Vol 16 (11) ◽  
pp. 431 ◽  
Author(s):  
Rosa Vitale ◽  
Enrico D'Aniello ◽  
Stefania Gorbi ◽  
Andrea Martella ◽  
Cristoforo Silvestri ◽  
...  
Keyword(s):  
Native Species ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Invasion Biology ◽  
In Vitro Assays ◽  
Bioactive Metabolites ◽  
Peroxisome Proliferator ◽  
Invasive Pests

Although the chemical warfare between invasive and native species has become a central problem in invasion biology, the molecular mechanisms by which bioactive metabolites from invasive pests influence local communities remain poorly characterized. This study demonstrates that the alkaloid caulerpin (CAU)—a bioactive component of the green alga Caulerpa cylindracea that has invaded the entire Mediterranean basin—is an agonist of peroxisome proliferator-activated receptors (PPARs). Our interdisciplinary study started with the in silico prediction of the ligand-protein interaction, which was then validated by in vivo, ex vivo and in vitro assays. On the basis of these results, we candidate CAU as a causal factor of the metabolic and behavioural disorders observed in Diplodus sargus, a native edible fish of high ecological and commercial relevance, feeding on C. cylindracea. Moreover, given the considerable interest in PPAR activators for the treatment of relevant human diseases, our findings are also discussed in terms of a possible nutraceutical/pharmacological valorisation of the invasive algal biomasses, supporting an innovative strategy for conserving biodiversity as an alternative to unrealistic campaigns for the eradication of invasive pests.

scholarly journals Microbial enzymes induce colitis by reactivating triclosan in the mouse gastrointestinal tract

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Jianan Zhang ◽  
Morgan E. Walker ◽  
Katherine Z. Sanidad ◽  
Hongna Zhang ◽  
Yanshan Liang ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Metabolic Activation ◽  
Consumer Products ◽  
Environmental Chemicals ◽  
Intestinal Microbes ◽  
Targeted Inhibition

AbstractEmerging research supports that triclosan (TCS), an antimicrobial agent found in thousands of consumer products, exacerbates colitis and colitis-associated colorectal tumorigenesis in animal models. While the intestinal toxicities of TCS require the presence of gut microbiota, the molecular mechanisms involved have not been defined. Here we show that intestinal commensal microbes mediate metabolic activation of TCS in the colon and drive its gut toxicology. Using a range of in vitro, ex vivo, and in vivo approaches, we identify specific microbial β-glucuronidase (GUS) enzymes involved and pinpoint molecular motifs required to metabolically activate TCS in the gut. Finally, we show that targeted inhibition of bacterial GUS enzymes abolishes the colitis-promoting effects of TCS, supporting an essential role of specific microbial proteins in TCS toxicity. Together, our results define a mechanism by which intestinal microbes contribute to the metabolic activation and gut toxicity of TCS, and highlight the importance of considering the contributions of the gut microbiota in evaluating the toxic potential of environmental chemicals.

scholarly journals Altered miRNA and mRNA Expression in Sika Deer Skeletal Muscle with Age

Genes ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 172
Author(s):  
Boyin Jia ◽  
Yuan Liu ◽  
Qining Li ◽  
Jiali Zhang ◽  
Chenxia Ge ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Growth And Development ◽  
Molecular Mechanisms ◽  
Muscle Development ◽  
Sika Deer ◽  
De Novo ◽  
Expression Profiles ◽  
Differentially Expressed ◽  
Growth Development ◽  
Development And Aging

Studies of the gene and miRNA expression profiles associated with the postnatal late growth, development, and aging of skeletal muscle are lacking in sika deer. To understand the molecular mechanisms of the growth and development of sika deer skeletal muscle, we used de novo RNA sequencing (RNA-seq) and microRNA sequencing (miRNA-seq) analyses to determine the differentially expressed (DE) unigenes and miRNAs from skeletal muscle tissues at 1, 3, 5, and 10 years in sika deer. A total of 51,716 unigenes, 171 known miRNAs, and 60 novel miRNAs were identified based on four mRNA and small RNA libraries. A total of 2,044 unigenes and 11 miRNAs were differentially expressed between adolescence and juvenile sika deer, 1,946 unigenes and 4 miRNAs were differentially expressed between adult and adolescent sika deer, and 2,209 unigenes and 1 miRNAs were differentially expressed between aged and adult sika deer. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that DE unigenes and miRNA were mainly related to energy and substance metabolism, processes that are closely associate with the growth, development, and aging of skeletal muscle. We also constructed mRNA–mRNA and miRNA–mRNA interaction networks related to the growth, development, and aging of skeletal muscle. The results show that mRNA (Myh1, Myh2, Myh7, ACTN3, etc.) and miRNAs (miR-133a, miR-133c, miR-192, miR-151-3p, etc.) may play important roles in muscle growth and development, and mRNA (WWP1, DEK, UCP3, FUS, etc.) and miRNAs (miR-17-5p, miR-378b, miR-199a-5p, miR-7, etc.) may have key roles in muscle aging. In this study, we determined the dynamic miRNA and unigenes transcriptome in muscle tissue for the first time in sika deer. The age-dependent miRNAs and unigenes identified will offer insights into the molecular mechanism underlying muscle development, growth, and maintenance and will also provide valuable information for sika deer genetic breeding.

scholarly journals TGF-β1 facilitates cell–cell communication in osteocytes via connexin43- and pannexin1-dependent gap junctions

2019 ◽  
Vol 5 (1) ◽  
Author(s):  
Wenjing Liu ◽  
Demao Zhang ◽  
Xin Li ◽  
Liwei Zheng ◽  
Chen Cui ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Lucifer Yellow ◽  
Cell Communication ◽  
Deep Understanding ◽  
Gap Junctional Intercellular Communication ◽  
Tgf Β1 ◽  
Cell Cell

Abstract Connexins and pannexins are two families of channel forming proteins that are able to pass small molecules to achieve communication between cells. While connexins have been recognized to mediate gap junctional intercellular communication (GJIC), pannexins are far less known. Our previous study reported the potential role of TGF-β1 in mediating of connexins in osteocytes in vitro. Herein, we aimed to elucidate the influence of TGF-β1 on cell–cell communication based on gap junctions assembled by connexins and pannexins in vitro and ex vivo. We first showed that TGF-β1 positively affected the elongation of dendritic processes of osteocytes. Our data indicated that TGF-β1 increased expressions of connexin43 (Cx43) and pannexin1 (panx1), which are indispensable for hemichannel formation in gap junctions, in osteocytes in vitro and ex vivo. TGF-β1 enhanced gap junction formation and impacted cell–cell communication in living osteocytes, as indicated by the scrape loading and Lucifer yellow transfer assays. TGF-β1 enhanced the expressions of Cx43 and panx1 via activation of ERK1/2 and Smad3/4 signalling. The TGF-β1-restored expressions of Cx43 and panx1 in osteocytes in the presence of an ERK inhibitor, U0126, further demonstrated the direct participation of Smad3/4 signalling. TGF-β1 increased the accumulation of Smad3 in the nuclear region (immunofluorescence assay) and promoted the enrichment of Smad3 at the binding sites of the promoters of Gja1 (Cx43) and Panx1 (ChIP assay), thereby initiating the enhanced gene expression. These results provide a deep understanding of the molecular mechanisms involved in the modulation of cell–cell communication in osteocytes induced by TGF-β1.

Sign in / Sign up

Export Citation Format

Share Document