Mechanical loading of bioengineered skeletal muscle in vitro recapitulates gene expression signatures of resistance exercise in vivo

Overloaded skeletal muscle undergoes dramatic shifts in gene expression, which alter both the phenotype and mass. Molecular biology techniques employing both in vivo and in vitro hypertrophy models have demonstrated that mechanical forces can alter skeletal muscle gene regulation. This review's purpose is to support integrin-mediated signaling as a candidate for mechanical load-induced hypertrophy. Research quantifying components of the integrin-signaling pathway in overloaded skeletal muscle have been integrated with knowledge regarding integrins role during development and cardiac hypertrophy, with the hope of demonstrating the pathway's importance. The role of integrin signaling as an integrator of mechanical forces and growth factor signaling during hypertrophy is discussed. Specific components of integrin signaling, including focal adhesion kinase and low-molecular-weight GTPase Rho are mentioned as downstream targets of this signaling pathway. There is a need for additional mechanistic studies capable of providing a stronger linkage between integrin-mediated signaling and skeletal muscle hypertrophy; however, there appears to be abundant justification for this type of research.

Download Full-text

Mechanical loading of tissue engineered skeletal muscle prevents dexamethasone induced myotube atrophy

Journal of Muscle Research and Cell Motility ◽

10.1007/s10974-020-09589-0 ◽

2020 ◽

Author(s):

Kathryn W. Aguilar-Agon ◽

Andrew J. Capel ◽

Jacob W. Fleming ◽

Darren J. Player ◽

Neil R. W. Martin ◽

...

Keyword(s):

Skeletal Muscle ◽

Mechanical Loading ◽

Muscle Atrophy ◽

Mechanical Load ◽

3D Models ◽

Skeletal Muscle Atrophy ◽

Treatment Modalities ◽

Murine Cell Line

Abstract Skeletal muscle atrophy as a consequence of acute and chronic illness, immobilisation, muscular dystrophies and aging, leads to severe muscle weakness, inactivity and increased mortality. Mechanical loading is thought to be the primary driver for skeletal muscle hypertrophy, however the extent to which mechanical loading can offset muscle catabolism has not been thoroughly explored. In vitro 3D-models of skeletal muscle provide a controllable, high throughput environment and mitigating many of the ethical and methodological constraints present during in vivo experimentation. This work aimed to determine if mechanical loading would offset dexamethasone (DEX) induced skeletal muscle atrophy, in muscle engineered using the C2C12 murine cell line. Mechanical loading successfully offset myotube atrophy and functional degeneration associated with DEX regardless of whether the loading occurred before or after 24 h of DEX treatment. Furthermore, mechanical load prevented increases in MuRF-1 and MAFbx mRNA expression, critical regulators of muscle atrophy. Overall, we demonstrate the application of tissue engineered muscle to study skeletal muscle health and disease, offering great potential for future use to better understand treatment modalities for skeletal muscle atrophy.

Download Full-text

Accelerated Response of the myogenin Gene to Denervation in Mutant Mice Lacking Phosphorylation of Myogenin at Threonine 87

Molecular and Cellular Biology ◽

10.1128/mcb.24.5.1983-1989.2004 ◽

2004 ◽

Vol 24 (5) ◽

pp. 1983-1989 ◽

Cited By ~ 11

Author(s):

Chris S. Blagden ◽

Larry Fromm ◽

Steven J. Burden

Keyword(s):

Gene Expression ◽

Skeletal Muscle ◽

Electrical Activity ◽

Muscle Development ◽

Mutant Mice ◽

Threonine Residue ◽

E Boxes ◽

Bhlh Proteins

ABSTRACT Gene expression in skeletal muscle is regulated by a family of myogenic basic helix-loop-helix (bHLH) proteins. The binding of these bHLH proteins, notably MyoD and myogenin, to E-boxes in their own regulatory regions is blocked by protein kinase C (PKC)-mediated phosphorylation of a single threonine residue in their basic region. Because electrical stimulation increases PKC activity in skeletal muscle, these data have led to an attractive model suggesting that electrical activity suppresses gene expression by stimulating phosphorylation of this critical threonine residue in myogenic bHLH proteins. We show that electrical activity stimulates phosphorylation of myogenin at threonine 87 (T87) in vivo and that calmodulin-dependent kinase II (CaMKII), as well as PKC, catalyzes this reaction in vitro. We find that phosphorylation of myogenin at T87 is dispensable for skeletal muscle development. We show, however, that the decrease in myogenin (myg) expression following innervation is delayed and that the increase in expression following denervation is accelerated in mutant mice lacking phosphorylation of myogenin at T87. These data indicate that two distinct innervation-dependent mechanisms restrain myogenin activity: an inactivation mechanism mediated by phosphorylation of myogenin at T87, and a second, novel regulatory mechanism that regulates myg gene activity independently of T87 phosphorylation.

Download Full-text

PL - 033 A translational model of muscle protein synthetic bioactivity in vitro, ex vivo and in vivo

Exercise Biochemistry Review ◽

10.14428/ebr.v1i1.8513 ◽

2018 ◽

Vol 1 (1) ◽

Cited By ~ 1

Author(s):

Brian Carson ◽

Robert Davies ◽

Joseph Bass ◽

Catherine Norton ◽

Bijal Patel ◽

...

Keyword(s):

Skeletal Muscle ◽

Stable Isotope ◽

Resistance Exercise ◽

Ex Vivo ◽

Muscle Protein ◽

Muscle Growth ◽

Translational Model ◽

Nutrient Feeding

Objective The aim of this research was the development and validation of a translational model for the evaluation of exercise and nutrient stimulated muscle protein synthesis (MPS). To achieve this overall aim, three primary objectives had to be realised: (i) Development of an in vitro skeletal muscle cell bioassay to measure muscle growth and MPS; (ii) Development of an ex vivo model to evaluate the humoral effect on MPS in response to nutrient feeding and exercise; (iii) Use of a stable isotope technique to evaluate MPS in response to nutrient feeding and exercise in vivo. Methods To develop a novel in vitro skeletal muscle cell bioassay to measure muscle growth and MPS, C2C12 myoblasts were proliferated and subsequently differentiated to myotubes over 8 days in DMEM (2% HS). Changes in cell behavior and adhesion properties were monitored by measuring impedance via interdigitated microelectrodes using the xCELLigence system. MPS was measured by puromycin incorporation using the SUnSET technique, intracellular signalling measured by western blot, and myotube thickness by microscopy. To demonstrate the capability to monitor nutrient regulation of muscle growth, media was conditioned with a known potent regulator of MPS (leucine) in a dose response experiment (0.20 - 2.0 mM). To establish the ability of the bioassay to measure the humoral effect of MPS in response to feeding and exercise, media was conditioned by ex vivo human serum from fasted, rested, fed (protein and isonitrogenous non-essential amino acid (NEAA) control) and post-exercise conditions. To evaluate MPS in response to nutrient feeding and exercise in vivo, acute MPS (5 h) was assessed by measuring stable isotope deuterium oxide (D2O) incorporation into m. vastus lateralis skeletal muscle following consumption of either a Whey Protein (WP) or an isonitrogenous NEAA control combined with resistance exercise in resistance trained males. Results In vitro experiments observed a dose-response effect with a 32 % increase in cell index and a 27 % increase in cell thickness after 2 h in the presence of 2.0 mM leucine when compared with control myotubes. Ex vivo serum following ingestion of NEAA had no effect on protein signalling or MPS whereas WP fed serum significantly increased mTOR, P70S6K and 4E-BP1 phosphorylation (p<0.01, p<0.05) compared to fasted serum. Furthermore, the effect of WP fed serum on protein signalling and MPS was significantly increased (p<0.01, p<0.05) compared to NEAA fed serum. Ex vivo human serum following resistance exercise was also increased MPS (29 %) and phosphorylation of mTOR (6 %), p70S6K (12 %) and 4EBP1 (7 %), compared with resting serum. These ex vivo/in vitro findings translated to the in vivo model as myofibrillar fractional synthetic rates (myoFSR) (Basal 0.068±0.002%h-1 vs. WP 0.084±0.006 %h-1, p=0.033) and absolute synthetic rates (ASR) (Basal 10.34±1.01 vs. WP 13.18±0.71 g.day-1, p=0.026) were increased from basal levels only when resistance exercise was combined with WP ingestion and not the NEAA control (NEAA MPS 0.072±0.004%h-1, NEAA ASR 10.23±0.80 g.day-1). Thus, ingestion of WP in combination with resistance training augments acute MPS responses in resistance trained young men. Conclusions We have developed a translational model of muscle protein synthetic bioactivity using in vitro, ex vivo and in vivo methodologies. We have shown that we can impact MPS in vitro using ex vivo human serum to condition media, that MPS in vitro is differentially regulated by ex vivo serum containing bioactive WP compared to a non-bioactive NEAA control, and that this tranlates for resistance exercise combined with WP in humans when MyoFSR is measured using stable isotope technology. These experiments demonstrate that ex vivo/in vitro experiments translate to the in vivo model and these methods can be used to inform both exercise and nutrient human interventions.

Download Full-text

Thyroid hormone receptors are down-regulated in skeletal muscle of patients with non-thyroidal illness syndrome secondary to non-septic shock

Acta Endocrinologica ◽

10.1530/eje-10-0376 ◽

2010 ◽

Vol 163 (5) ◽

pp. 765-773 ◽

Cited By ~ 10

Author(s):

J Lado-Abeal ◽

A Romero ◽

I Castro-Piedras ◽

A Rodriguez-Perez ◽

J Alvarez-Escudero

Keyword(s):

Gene Expression ◽

Skeletal Muscle ◽

Septic Shock ◽

Thyroid Hormone ◽

Hormone Receptors ◽

Serum Samples ◽

Pathway Activation ◽

Gene And Protein Expression

AimNon-thyroidal illness syndrome (NTIS) is related to changes in thyroid hormone (TH) physiology. Skeletal muscle (SM) plays a major role in metabolism, and TH regulates SM phenotype and metabolism. We aimed to characterize the SM of non-septic shock NTIS patients in terms of: i) expression of genes and proteins involved in TH metabolism and actions; and ii) NFKB's pathway activation, a responsible factor for some of the phenotypic changes in NTIS. We also investigated whether the patient's serum can induce in vitro the effects observed in vivo.MethodsSerum samples and SM biopsies from 14 patients with non-septic shock NTIS and 11 controls. Gene and protein expression and NFKB1 activation were analyzed by quantitative PCR and immunoblotting. Human SM cell (HSkMC) cultures to investigate the effects of patient's serum on TH action mediators.ResultsPatients with non-septic shock NTIS showed higher levels of pro-inflammatory cytokines than controls. Expression of TRβ (THRB), TRα1 (THRA), and retinoid X receptor γ (RXRG) was decreased in NTIS patients. RXRA gene expression was higher, but its protein was lower in NTIS than controls, suggesting the existence of a post-transcriptional mechanism that down-regulates protein levels. NFKB1 pathway activation was not different between NTIS and control patients. HSkMC incubated with patient's serum increased TH receptor and RXRG gene expression after 48 h.ConclusionsPatients with non-septic shock NTIS showed decreased expression of TH receptors and RXRs, which were not related to increased activation of the NFKB1 pathway. These findings could not be replicated in cultures of HSkMCs incubated in the patient's serum.

Download Full-text

Cellular mechanisms regulating protein synthesis and skeletal muscle hypertrophy in animals

Journal of Applied Physiology ◽

10.1152/japplphysiol.91355.2008 ◽

2009 ◽

Vol 106 (4) ◽

pp. 1367-1373 ◽

Cited By ~ 110

Author(s):

Mitsunori Miyazaki ◽

Karyn A. Esser

Keyword(s):

Skeletal Muscle ◽

Cell Culture ◽

Protein Synthesis ◽

Mechanical Loading ◽

Muscle Hypertrophy ◽

Mammalian Target Of Rapamycin ◽

Cellular Mechanisms ◽

Skeletal Muscle Hypertrophy

Growth and maintenance of skeletal muscle mass is critical for long-term health and quality of life. Skeletal muscle is a highly adaptable tissue with well-known sensitivities to environmental cues such as growth factors, cytokines, nutrients, and mechanical loading. All of these factors act at the level of the cell and signal through pathways that lead to changes in phenotype through multiple mechanisms. In this review, we discuss the animal and cell culture models used and the signaling mechanisms identified in understanding regulation of protein synthesis in response to mechanical loading/resistance exercise. Particular emphasis has been placed on 1) alterations in mechanical loading and regulation of protein synthesis in both in vivo animal studies and in vitro cell culture studies and 2) upstream mediators regulating mammalian target of rapamycin signaling and protein synthesis during skeletal muscle hypertrophy.

Download Full-text

Myogenin is required for assembly of the transcription machinery on muscle genes during skeletal muscle differentiation

PLoS ONE ◽

10.1371/journal.pone.0245618 ◽

2021 ◽

Vol 16 (1) ◽

pp. e0245618

Author(s):

Abhinav Adhikari ◽

William Kim ◽

Judith Davie

Keyword(s):

Gene Expression ◽

Skeletal Muscle ◽

Gene Activation ◽

Gene Promoters ◽

Muscle Gene Expression ◽

Transcription Machinery ◽

Muscle Genes ◽

Muscle Gene

Skeletal muscle gene expression is governed by the myogenic regulatory family (MRF) which includes MyoD (MYOD1) and myogenin (MYOG). MYOD1 and MYOG are known to regulate an overlapping set of muscle genes, but MYOD1 cannot compensate for the absence of MYOG in vivo. In vitro, late muscle genes have been shown to be bound by both factors, but require MYOG for activation. The molecular basis for this requirement was unclear. We show here that MYOG is required for the recruitment of TBP and RNAPII to muscle gene promoters, indicating that MYOG is essential in assembling the transcription machinery. Genes regulated by MYOD1 and MYOG include genes required for muscle fusion, myomaker and myomerger, and we show that myomaker is fully dependent on activation by MYOG. We also sought to determine the role of MYOD1 in MYOG dependent gene activation and unexpectedly found that MYOG is required to maintain Myod1 expression. However, we also found that exogenous MYOD1 was unable to compensate for the loss of Myog and activate muscle gene expression. Thus, our results show that MYOD1 and MYOG act in a feed forward loop to maintain each other’s expression and also show that it is MYOG, and not MYOD1, that is required to load TBP and activate gene expression on late muscle gene promoters bound by both factors.

Download Full-text

In Vivo and In Vitro Mechanical Loading of Mouse Achilles Tendons and Tenocytes—A Pilot Study

International Journal of Molecular Sciences ◽

10.3390/ijms21041313 ◽

2020 ◽

Vol 21 (4) ◽

pp. 1313

Author(s):

Viviane Fleischhacker ◽

Franka Klatte-Schulz ◽

Susann Minkwitz ◽

Aysha Schmock ◽

Maximilian Rummler ◽

...

Keyword(s):

Gene Expression ◽

Pilot Study ◽

Mechanical Loading ◽

Cell Shape ◽

Compressive Loading ◽

Collagen Type I ◽

Type I ◽

The Impact

Mechanical force is a key factor for the maintenance, adaptation, and function of tendons. Investigating the impact of mechanical loading in tenocytes and tendons might provide important information on in vivo tendon mechanobiology. Therefore, the study aimed at understanding if an in vitro loading set up of tenocytes leads to similar regulations of cell shape and gene expression, as loading of the Achilles tendon in an in vivo mouse model. In vivo: The left tibiae of mice (n = 12) were subject to axial cyclic compressive loading for 3 weeks, and the Achilles tendons were harvested. The right tibiae served as the internal non-loaded control. In vitro: tenocytes were isolated from mice Achilles tendons and were loaded for 4 h or 5 days (n = 6 per group) based on the in vivo protocol. Histology showed significant differences in the cell shape between in vivo and in vitro loading. On the molecular level, quantitative real-time PCR revealed significant differences in the gene expression of collagen type I and III and of the matrix metalloproteinases (MMP). Tendon-associated markers showed a similar expression profile. This study showed that the gene expression of tendon markers was similar, whereas significant changes in the expression of extracellular matrix (ECM) related genes were detected between in vivo and in vitro loading. This first pilot study is important for understanding to which extent in vitro stimulation set-ups of tenocytes can mimic in vivo characteristics.

Download Full-text

Regulation of SREBP1c Gene Expression in Skeletal Muscle: Role of Retinoid X Receptor/Liver X Receptor and Forkhead-O1 Transcription Factor

Endocrinology ◽

10.1210/en.2007-1461 ◽

2008 ◽

Vol 149 (5) ◽

pp. 2293-2305 ◽

Cited By ~ 47

Author(s):

Yasutomi Kamei ◽

Shinji Miura ◽

Takayoshi Suganami ◽

Fumiko Akaike ◽

Sayaka Kanai ◽

...

Keyword(s):

Gene Expression ◽

Skeletal Muscle ◽

Transcription Factor ◽

Transgenic Mice ◽

Regulatory Element ◽

Liver X Receptor ◽

Retinoid X Receptor ◽

Dominant Negative

Sterol regulatory element binding protein 1c (SREBP1c) is a master regulator of lipogenic gene expression in liver and adipose tissue, where its expression is regulated by a heterodimer of nuclear receptor-type transcription factors retinoid X receptor-α (RXRα) and liver X receptor-α (LXRα). Despite the potential importance of SREBP1c in skeletal muscle, little is known about the regulation of SREBP1c in that setting. Here we report that gene expression of RXRγ is markedly decreased by fasting and is restored by refeeding in mouse skeletal muscle, in parallel with changes in gene expression of SREBP1c. RXRγ or RXRα, together with LXRα, activate the SREBP1c promoter in vitro. Moreover, transgenic mice overexpressing RXRγ specifically in skeletal muscle showed increased gene expression of SREBP1c with increased triglyceride content in their skeletal muscles. In contrast, transgenic mice overexpressing the dominant-negative form of RXRγ showed decreased SREBP1c gene expression. The expression of Forkhead-O1 transcription factor (FOXO1), which can suppress the function of multiple nuclear receptors, is negatively correlated to that of SREBP1c in skeletal muscle during nutritional change. Moreover, transgenic mice overexpressing FOXO1 specifically in skeletal muscle exhibited decreased gene expression of both RXRγ and SREBP1c. In addition, FOXO1 suppressed RXRα/LXRα-mediated SREBP1c promoter activity in vitro. These findings provide in vivo and in vitro evidence that RXR/LXR up-regulates SREBP1c gene expression and that FOXO1 antagonizes this effect of RXR/LXR in skeletal muscle.

Download Full-text