The lumbrical muscle: a novel in situ system to evaluate adult skeletal muscle proteolysis and anticatabolic drugs for therapeutic purposes

The molecular regulation of skeletal muscle proteolysis and the pharmacological screening of anticatabolic drugs have been addressed by measuring tyrosine release from prepubertal rat skeletal muscles, which are thin enough to allow adequate in vitro diffusion of oxygen and substrates. However, the use of muscle at accelerated prepubertal growth has limited the analysis of adult muscle proteolysis or that associated with aging and neurodegenerative diseases. Here we established the adult rat lumbrical muscle (4/hindpaw; 8/rat) as a new in situ experimental model for dynamic measurement of skeletal muscle proteolysis. By incubating lumbrical muscles attached to their individual metatarsal bones in Tyrode solution, we showed that the muscle proteolysis rate of adult and aged rats (3–4 to 24 mo old) is 45–25% of that in prepubertal animals (1 mo old), which makes questionable the usual extrapolation of proteolysis from prepubertal to adult/senile muscles. While acute mechanical injury or 1- to 7-day denervation increased tyrosine release from adult lumbrical muscle by up to 60%, it was reduced by 20–28% after 2-h incubation with β-adrenoceptor agonists, forskolin or phosphodiesterase inhibitor IBMX. Using inhibitors of 26S-proteasome (MG132), lysosome (methylamine), or calpain (E64/leupeptin) systems, we showed that ubiquitin-proteasome is accountable for 40–50% of total lumbrical proteolysis of adult, middle-aged, and aged rats. In conclusion, the lumbrical model allows the analysis of muscle proteolysis rate from prepubertal to senile rats. By permitting eight simultaneous matched measurements per rat, the new model improves similar protocols performed in paired extensor digitorum longus (EDL) muscles from prepubertal rats, optimizing the pharmacological screening of drugs for anticatabolic purposes.

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Visualizing miRNAs in adult skeletal muscle by hybridization in situ

Protocol Exchange ◽

10.1038/nprot.2006.136 ◽

2006 ◽

Author(s):

Anna Polesskaya

Keyword(s):

Skeletal Muscle ◽

Adult Skeletal Muscle ◽

Hybridization In Situ

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The cell adhesion molecule M-cadherin is specifically expressed in developing and regenerating, but not denervated skeletal muscle

Development ◽

10.1242/dev.117.4.1409 ◽

1993 ◽

Vol 117 (4) ◽

pp. 1409-1420 ◽

Cited By ~ 5

Author(s):

R. Moore ◽

F.S. Walsh

Keyword(s):

Skeletal Muscle ◽

Tissue Distribution ◽

Cell Adhesion Molecule ◽

Muscle Denervation ◽

Regulatory Factor ◽

Myogenic Regulatory Factor ◽

Adult Skeletal Muscle ◽

Specific Tissue ◽

Alpha Actin

The spatiotemporal distribution of M-cadherin mRNA has been determined by in situ hybridization in the mouse embryo and in adult skeletal muscle following experimental regeneration and denervation. M-cadherin mRNA is highly tissue specific and is found only in developing skeletal muscle. In contrast, N-cadherin mRNA has a broader tissue distribution in the embryo, being found on both neural elements and skeletal and cardiac muscle. M-cadherin is expressed in the myotomes shortly after they form, along with the myogenic regulatory factor myogenin. M-cadherin is expressed in muscles derived from the myotomes and is detected in forelimb bud precursor cells at embryonic day 11.5. In the latter case M-cadherin expression appears co-ordinately with that of myogenin and cardiac alpha-actin. Shortly before birth, M-cadherin expression is down regulated. M-cadherin can, however, be re-expressed following experimental regeneration of skeletal muscle. Here M-cadherin is transiently expressed on regenerating myoblasts but not myotubes. Following muscle denervation no evidence was found for re-expression of M-cadherin under conditions where there was strong expression of the nicotinic acetylcholine receptor on myofibres. The highly specific tissue distribution and unique developmental profile distinguishes M-cadherin from other cadherins and suggests a role in cell surface events during early myogenesis.

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Effects of aging and exercise training on spinotrapezius muscle microvascular Po2 dynamics and vasomotor control

Journal of Applied Physiology ◽

10.1152/japplphysiol.01084.2010 ◽

2011 ◽

Vol 110 (3) ◽

pp. 695-704 ◽

Cited By ~ 19

Author(s):

Danielle J. McCullough ◽

Robert T. Davis ◽

James M. Dominguez ◽

John N. Stabley ◽

Christian S. Bruells ◽

...

Keyword(s):

Skeletal Muscle ◽

Blood Flow ◽

Exercise Training ◽

Sodium Nitroprusside ◽

Vascular Function ◽

Treadmill Running ◽

Aged Rats ◽

Phosphorescence Quenching

With advancing age, there is a reduction in exercise tolerance, resulting, in part, from a perturbed ability to match O2 delivery to uptake within skeletal muscle. In the spinotrapezius muscle (which is not recruited during incline treadmill running) of aged rats, we tested the hypotheses that exercise training will 1) improve the matching of O2 delivery to O2 uptake, evidenced through improved microvascular Po2 (PmO2), at rest and throughout the contractions transient; and 2) enhance endothelium-dependent vasodilation in first-order arterioles. Young (Y, ∼6 mo) and aged (O, >24 mo) Fischer 344 rats were assigned to control sedentary (YSED; n = 16, and OSED; n = 15) or exercise-trained (YET; n = 14, and OET; n = 13) groups. Spinotrapezius blood flow (via radiolabeled microspheres) was measured at rest and during exercise. Phosphorescence quenching was used to quantify PmO2 in vivo at rest and across the rest-to-twitch contraction (1 Hz, 5 min) transition in the spinotrapezius muscle. In a follow-up study, vasomotor responses to endothelium-dependent (acetylcholine) and -independent (sodium nitroprusside) stimuli were investigated in vitro. Blood flow to the spinotrapezius did not increase above resting values during exercise in either young or aged groups. Exercise training increased the precontraction baseline PmO2 (OET 37.5 ± 3.9 vs. OSED 24.7 ± 3.6 Torr, P < 0.05); the end-contracting PmO2 and the time-delay before PmO2 fell in the aged group but did not affect these values in the young. Exercise training improved maximal vasodilation in aged rats to acetylcholine (OET 62 ± 16 vs. OSED 27 ± 16%) and to sodium nitroprusside in both young and aged rats. Endurance training of aged rats enhances the PmO2 in a nonrecruited skeletal muscle and is associated with improved vascular smooth muscle function. These data support the notion that improvements in vascular function with exercise training are not isolated to the recruited muscle.

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A Nonerythroid Isoform of Protein 4.1R Interacts with Components of the Contractile Apparatus in Skeletal Myofibers

Molecular Biology of the Cell ◽

10.1091/mbc.11.11.3805 ◽

2000 ◽

Vol 11 (11) ◽

pp. 3805-3817 ◽

Cited By ~ 29

Author(s):

Aikaterini Kontrogianni-Konstantopoulos ◽

Shu-Ching Huang ◽

Edward J. Benz

Keyword(s):

Skeletal Muscle ◽

Protein S ◽

Binding Assays ◽

Adult Skeletal Muscle ◽

Exon 2 ◽

Alternatively Spliced ◽

Translation Initiation Codon ◽

Protein 4.1R

The ∼80-kDa erythroid 4.1R protein is a major component of the erythrocyte cytoskeleton, where it links transmembrane proteins to the underlying spectrin/actin complexes. A diverse collection of 4.1R isoforms has been described in nonerythroid cells, ranging from ∼30 to ∼210 kDa. In the current study, we identified the number and primary structure of 4.1R isoforms expressed in adult skeletal muscle and characterized the localization patterns of 4.1R message and protein. Skeletal muscle 4.1R appears to originate solely from the upstream translation initiation codon (AUG-1) residing in exon 2′. Combinations of alternatively spliced downstream exons generate an array of distinct 4.1R spliceoforms. Two major isoform classes of ∼105/110 and ∼135 kDa are present in muscle homogenates. 4.1R transcripts are distributed in highly ordered signal stripes, whereas 4.1R protein(s) decorate the sarcoplasm in transverse striations that are in register with A-bands. An ∼105/110-kDa 4.1R isoform appears to occur in vivo in a supramolecular complex with major sarcomeric proteins, including myosin, α-actin, and α-tropomyosin. In vitro binding assays showed that 4.1R may interact directly with the aforementioned contractile proteins through its 10-kDa domain. All of these observations suggest a topological model whereby 4.1R may play a scaffolding role by anchoring the actomyosin myofilaments and possibly modulating their displacements during contraction/relaxation.

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Molecular Targets of Androgen Signaling that Characterize Skeletal Muscle Recovery and Regeneration

Nuclear Receptor Signaling ◽

10.1621/nrs.13005 ◽

2015 ◽

Vol 13 (1) ◽

pp. nrs.13005 ◽

Cited By ~ 11

Author(s):

James G. MacKrell ◽

Benjamin C. Yaden ◽

Heather Bullock ◽

Keyue Chen ◽

Pamela Shetler ◽

...

Keyword(s):

Skeletal Muscle ◽

Molecular Targets ◽

Gene Signature ◽

Muscle Recovery ◽

In Vivo Models ◽

Adult Skeletal Muscle ◽

Injury Model ◽

Androgen Regulation

The high regenerative capacity of adult skeletal muscle relies on a self-renewing depot of adult stem cells, termed muscle satellite cells (MSCs). Androgens, known mediators of overall body composition and specifically skeletal muscle mass, have been shown to regulate MSCs. The possible overlapping function of androgen regulation of muscle growth and MSC activation has not been carefully investigated with regards to muscle regeneration. Therefore, the aim of this study was to examine coinciding androgen-mediated genetic changes in an in vitro MSC model and clinically relevant in vivo models. A gene signature was established via microarray analysis for androgen-mediated MSC engagement and highlighted several markers including follistatin (FST), IGF-1, C-X-C chemokine receptor 4 (CXCR4), hepatocyte growth factor (HGF) and glucocorticoid receptor (GR/Nr3c1). In an in vivo muscle atrophy model, androgen re-supplementation significantly increased muscle size and expression of IGF-1, FST, and HGF, while significantly decreasing expression of GR. Biphasic gene expression profiles over the 7-day re-supplementation period identifed temporal androgen regulation of molecular targets involved in satellite cell engagement into myogenesis. In a muscle injury model, removal of androgens resulted in delayed muscle recovery and regeneration. Modifications in the androgen signaling gene signature, along with reduced Pax7 and MyoD expression, suggested that limited MSC activation and increased inflammation contributed to the delayed regeneration. However, enhanced MSC activation in the androgen-deplete mouse injury model was driven by an androgen receptor (AR) agonist. These results provide novel in vitro and in vivo evidence describing molecular targets of androgen signaling, while also increasing support for translational use of AR agonists in skeletal muscle recovery and regeneration.

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FGF-2–dependent signaling activated in aged human skeletal muscle promotes intramuscular adipogenesis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2021013118 ◽

2021 ◽

Vol 118 (37) ◽

pp. e2021013118 ◽

Cited By ~ 1

Author(s):

Sebastian Mathes ◽

Alexandra Fahrner ◽

Umesh Ghoshdastider ◽

Hannes A. Rüdiger ◽

Michael Leunig ◽

...

Keyword(s):

Skeletal Muscle ◽

Transcriptional Activation ◽

Human Skeletal Muscle ◽

Muscle Growth ◽

Muscle Cells ◽

Adeno Associated Virus ◽

Adult Skeletal Muscle

Aged skeletal muscle is markedly affected by fatty muscle infiltration, and strategies to reduce the occurrence of intramuscular adipocytes are urgently needed. Here, we show that fibroblast growth factor-2 (FGF-2) not only stimulates muscle growth but also promotes intramuscular adipogenesis. Using multiple screening assays upstream and downstream of microRNA (miR)-29a signaling, we located the secreted protein and adipogenic inhibitor SPARC to an FGF-2 signaling pathway that is conserved between skeletal muscle cells from mice and humans and that is activated in skeletal muscle of aged mice and humans. FGF-2 induces the miR-29a/SPARC axis through transcriptional activation of FRA-1, which binds and activates an evolutionary conserved AP-1 site element proximal in the miR-29a promoter. Genetic deletions in muscle cells and adeno-associated virus–mediated overexpression of FGF-2 or SPARC in mouse skeletal muscle revealed that this axis regulates differentiation of fibro/adipogenic progenitors in vitro and intramuscular adipose tissue (IMAT) formation in vivo. Skeletal muscle from human donors aged >75 y versus <55 y showed activation of FGF-2–dependent signaling and increased IMAT. Thus, our data highlights a disparate role of FGF-2 in adult skeletal muscle and reveals a pathway to combat fat accumulation in aged human skeletal muscle.

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MyoScreen, a High-Throughput Phenotypic Screening Platform Enabling Muscle Drug Discovery

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555218761102 ◽

2018 ◽

Vol 23 (8) ◽

pp. 790-806 ◽

Cited By ~ 1

Author(s):

Joanne Young ◽

Yoran Margaron ◽

Mathieu Fernandes ◽

Eve Duchemin-Pelletier ◽

Joris Michaud ◽

...

Keyword(s):

Skeletal Muscle ◽

High Throughput ◽

Acetylcholine Receptors ◽

Calcium Release ◽

Screening Strategy ◽

Adult Skeletal Muscle ◽

New Chemical Entities ◽

Drug Treatments ◽

Screening Platform

Despite the need for more effective drug treatments to address muscle atrophy and disease, physiologically accurate in vitro screening models and higher information content preclinical assays that aid in the discovery and development of novel therapies are lacking. To this end, MyoScreen was developed: a robust and versatile high-throughput high-content screening (HT/HCS) platform that integrates a physiologically and pharmacologically relevant micropatterned human primary skeletal muscle model with a panel of pertinent phenotypic and functional assays. MyoScreen myotubes form aligned, striated myofibers, and they show nerve-independent accumulation of acetylcholine receptors (AChRs), excitation–contraction coupling (ECC) properties characteristic of adult skeletal muscle and contraction in response to chemical stimulation. Reproducibility and sensitivity of the fully automated MyoScreen platform are highlighted in assays that quantitatively measure myogenesis, hypertrophy and atrophy, AChR clusterization, and intracellular calcium release dynamics, as well as integrating contractility data. A primary screen of 2560 compounds to identify stimulators of myofiber regeneration and repair, followed by further biological characterization of two hits, validates MyoScreen for the discovery and testing of novel therapeutics. MyoScreen is an improvement of current in vitro muscle models, enabling a more predictive screening strategy for preclinical selection of the most efficacious new chemical entities earlier in the discovery pipeline process.

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Enhanced skeletal muscle arteriolar reactivity to ANG II after recovery from ischemic acute renal failure

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00269.2005 ◽

2005 ◽

Vol 289 (6) ◽

pp. R1770-R1776 ◽

Cited By ~ 10

Author(s):

David P. Basile ◽

Deborah L. Donohoe ◽

Shane A. Phillips ◽

Jefferson C. Frisbee

Keyword(s):

Skeletal Muscle ◽

Renal Failure ◽

Acute Renal Failure ◽

Pressor Response ◽

Gracilis Muscle ◽

Ang Ii ◽

Sprague Dawley

In addition to the long-term renal complications, previous studies suggested that after acute renal failure (ARF), rats manifest an increased pressor response to an overnight infusion of ANG II. The present study tested whether recovery from ARF results in alterations in sensitivity to the peripheral vasculature. ARF was induced in Sprague-Dawley rats by 45 min of bilateral renal ischemia and reperfusion. Animals were allowed to recover renal structure and function for 5–8 wk, after which the acute pressor responses to ANG II were evaluated either in vivo in in situ skeletal muscle arterioles or in isolated gracilis muscle arteries in vitro. Baseline arterial pressure was not different in ARF rats vs. sham-operated controls, although ARF rats exhibited an enhanced pressor response to bolus ANG II infusion compared with control rats. Steady-state plasma ANG II concentration and plasma renin activity were similar between ARF and control rats. Constrictor reactivity of in situ cremasteric arterioles from ARF rats was enhanced in response to increasing concentrations of ANG II; however, no difference was observed in arteriolar responses to elevated Po2, norepinephrine, acetylcholine, or sodium nitroprusside. Isolated gracilis muscle arteries from ARF rats also showed increased vasoconstriction in response to ANG II but not norepinephrine. In conclusion, recovery from ischemic ARF is not associated with hypertension but is associated with increased arteriolar constrictor reactivity to ANG II. Although the mechanisms of this altered responsiveness are unclear, such changes may relate, in part, to cardiovascular complications in patients with ARF and/or after renal transplant.

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Thrombospondin-4, an extracellular matrix protein expressed in the developing and adult nervous system promotes neurite outgrowth.

The Journal of Cell Biology ◽

10.1083/jcb.131.4.1083 ◽

1995 ◽

Vol 131 (4) ◽

pp. 1083-1094 ◽

Cited By ~ 102

Author(s):

S Arber ◽

P Caroni

Keyword(s):

Skeletal Muscle ◽

Extracellular Matrix ◽

Nervous System ◽

Neuromuscular Junction ◽

Neurite Outgrowth ◽

Adult Skeletal Muscle ◽

Wide Range ◽

Thrombospondin 4

Extracellular matrix (ECM) molecules are involved in multiple aspects of cell-to-cell signaling during development and in the adult. In nervous system development, specific recognition processes, e.g., during axonal pathfinding and synaptogenesis involve modulation and signaling by ECM components. Much less is known about their presence and possible roles in the adult nervous system. We now report that thrombospondin-4 (TSP-4), a recently discovered member of the TSP gene family is expressed by neurons, promotes neurite outgrowth, and accumulates at the neuromuscular junction and at certain synapse-rich structures in the adult. To search for muscle genes that may be involved in neuromuscular signaling, we isolated cDNAs induced in adult skeletal muscle by denervation. One of these cDNAs coded for the rat homologue of TSP-4. In skeletal muscle, it was expressed by muscle interstitial cells. The transcript was further detected in heart and in the developing and adult nervous system, where it was expressed by a wide range of neurons. An antiserum to the unique carboxyl-terminal end of the protein allowed to specifically detect TSP-4 in transfected cells in vitro and on cryostat sections in situ. TSP-4 associated with ECM structures in vitro and in vivo. In the adult, it accumulated at the neuromuscular junction and at synapse-rich structures in the cerebellum and retina. To analyze possible activities of TSP-4 towards neurons, we carried out coculture experiments with stably transfected COS cells and motor, sensory, or retina neurons. These experiments revealed that TSP-4 was a preferred substrate for these neurons, and promoted neurite outgrowth. The results establish TSP-4 as a neuronal ECM protein associated with certain synapse-rich structures in the adult. Its activity towards embryonic neurons in vitro and its distribution in vivo suggest that it may be involved in local signaling in the developing and adult nervous system.

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