Fast-twitch Muscle Research Articles

Low Intensity Vibration Treatment Reduces Myeloma Cell-Induced Osteoclast Formation By Inhibiting IL-6 and GFI-1 Production By MM Cells

Multiple myeloma (MM) bone disease is characterized by increased osteoclast-mediated resorption of bone adjacent to tumor growth. Fusion and activation of osteoclasts (OC), indicated by elevated tartrate-resistant acid phosphatase (TRAP) production, are induced by a myriad of inflammatory factors; e.g. TGF-β, growth factor independent 1 transcription factor (GFI-1), RANKL, and IL-6 that are produced by myeloma cells and cells in the marrow microenvironment. Current therapies reduce tumor burden and block osteoclast formation and activity, thereby decreasing bone destruction. However, MM bones rarely heal. While the anabolic effects of exercise on healthy bone are well-documented, patients with compromised skeletal tissue, such as in MM, may not be able to participate in regimented exercise for fear of developing a fracture. Low intensity vibrations (LIV) that deliver subtle mechanical signals, on the order of those induced by fast-twitch muscle fibers on bone, can enhance mesenchymal stem cell differentiation towards osteoblasts. LIV also significantly preserves trabecular bone while reducing tumor burden in femora of mice harboring U266 MM cells. We hypothesize that LIV is both anti-resorptive and anabolic, through differential effects on MM cells and bone cells. To test this hypothesis, we used a vertically oscillating, vibratory platform that delivered mechanical signals (<1 x Earth's gravitational acceleration; where 1g=9.8m/s2) to culture plates.5TGM1 murine MM cells (5x105 cells/well) were cultured in RPMI with 10% FCS and subjected to LIV or Sham-LIV. Each 6-well plate received either LIV (frequency=90Hertz, acceleration=0.3g) or Sham-LIV (SH; unpowered, static platform) twice per day for 20min per treatment for 48hrs. MM cell lysates were then collected using RIPA buffer. IL-6, GFI-1, and RANKL levels were determined via Western Blot using specific monoclonal antibodies. GAPDH was used as the loading control. Conditioned media (CM) were isolated from SH- and LIV-treated MM cultures. Purified primary murine OC precursors (non-adherent marrow mononuclear cells) were expanded with 10ng/mL of MCSF for 3 days and then these cells were isolated by trypsinization and replated in 12-well plates at a density of 5x105 cells/well in α-MEM, 10% FCS, and 30% v/v CM. All cultures were then incubated for 36hrs. OC number and morphology were confirmed via TRAP staining (counterstained with hematoxylin) of the cultures. TRAP+ OC's containing at least three nuclei were quantified in the cultures by microscopy.Expression levels of IL-6 and GFI-1 were 53% (p<0.01) and 50% (p<0.02) lower, respectively, in lysates from LIV-exposed 5TGM1 cells as compared to those that were SH-treated. Expression of RANKL was not significantly different between these groups. TRAP+ OC's were 61% (p<0.005) lower in LIV-treated cultures compared with SH cultures. The morphology of OC's exposed to LIV-conditioned media from MM cultures was distinct from control cultures, with fewer filopodia than OC's in cultures treated with SH-conditioned media. Based on these data we then examined the direct effect of LIV-treatment on committed OC-precursors treated with RANKL (50ng/mL) to induce formation of mature OC's. After 48hrs of LIV-treatment, committed OC-precursors had 36% (p<0.0001) fewer nuclei per OC than in SH-treated cultures. The cultures of committed OC-precursors directly treated with LIV had fewer filopodia in comparison to SH-treated cultures upon examination of OC morphology.These findings suggest that mechanical signals in the form of LIV may influence the secretion of IL-6 and GFI-1 by myeloma cells, which, in turn, can decrease the capacity of MM cells to induce OC formation. The morphology of OC's and expression of TRAP by OC's exposed to LIV further suggest a distinct mechanosensitive effect on osteoclast formation and fusion that may impact their bone resorbing capacity. Thus, LIV has a significant effect on the capacity of MM cells to secrete inflammatory cytokines as well as directly affect OC formation. These results further suggest that low intensity vibrations may be a useful, non-invasive approach for treating myeloma bone disease. DisclosuresRoodman:Amgen Denosumab Trial: Membership on an entity's Board of Directors or advisory committees.

Myosin-based regulation of twitch and tetanic contractions in mammalian skeletal muscle.

Time-resolved X-ray diffraction of isolated fast-twitch muscles of mice was used to show how structural changes in the myosin-containing thick filaments contribute to the regulation of muscle contraction, extending the previous focus on regulation by the actin-containing thin filaments. This study shows that muscle activation involves the following sequence of structural changes: thin filament activation, disruption of the helical array of myosin motors characteristic of resting muscle, release of myosin motor domains from the folded conformation on the filament backbone, and actin attachment. Physiological force generation in the 'twitch' response of skeletal muscle to single action potential stimulation is limited by incomplete activation of the thick filament and the rapid inactivation of both filaments. Muscle relaxation after repetitive stimulation is accompanied by a complete recovery of the folded motor conformation on the filament backbone but by incomplete reformation of the helical array, revealing a structural basis for post-tetanic potentiation in isolated muscles.

Sexual dimorphism in vascular ATP-sensitive K+ channel function supporting interstitial via convective and/or diffusive O2 transport.

Inhibition of pancreatic ATP-sensitive K+ (KATP ) channels is the intended effect of oral sulphonylureas to increase insulin release in diabetes. However, pertinent to off-target effects of sulphonylurea medication, sex differences in cardiac KATP channel function exist, whereas potential sex differences in vascular KATP channel function remain unknown. In the present study, we assessed vascular KATP channel function (topical glibenclamide superfused onto fast-twitch oxidative skeletal muscle) supporting blood flow and interstitial O2 delivery-utilization matching ( is) during twitch contractions in male, female during pro-oestrus and ovariectomized female (F+OVX) rats. Glibenclamide decreased blood flow (convective O2 transport) and interstitial in male and female, but not F+OVX, rats. Compared to males, females also demonstrated impaired diffusive O2 transport and a faster fall in interstitial . Our demonstration, in rats, that sex differences in vascular KATP channel function exist support the tentative hypothesis that oral sulphonylureas may exacerbate exercise intolerance and morbidity, especially in premenopausal females. Vascular ATP-sensitive K+ (KATP ) channels support skeletal muscle blood flow ( ), interstitial O2 delivery ( )-utilization ( ) matching (i.e. interstitial-myocyte O2 flux driving pressure; is) and exercise tolerance. Potential sex differences in skeletal muscle vascular KATP channel function remain largely unexplored. We hypothesized that local skeletal muscle KATP channel inhibition via glibenclamide superfusion (5mg kg-1 GLI; sulphonylurea diabetes medication) in anaesthetized female Sprague-Dawley rats, compared to males, would demonstrate greater reductions in contracting (1Hz, 7V, 180s) fast-twitch oxidative mixed gastrocnemius (97% type IIA+IID/X+IIB) (15μm microspheres) and is (phosphorescence quenching), resulting from more compromised convective ( ) and diffusive ( ) O2 conductances. Furthermore, these GLI-induced reductions in ovary-intact females measured during pro-oestrus would be diminished following ovariectomy (F+OVX). GLI similarly impaired mixed gastrocnemius in both males (↓28%) and females (↓33%, both P<0.032) via reduced (male: ↓31%, female: ↓35%, both P<0.020), (male: 5.6±0.5 vs. 4.0±0.5, female: 6.4±1.1 vs. 4.2±0.6mL O2 min-1 100g tissue-1 , P<0.022) and the resulting is, with females also demonstrating a reduced (0.40±0.07 vs. 0.30±0.04mL O2 min-1 100g tissue-1 , P<0.042) and a greater GLI-induced speeding of is fall (mean response time: Sex×Drug interaction, P=0.026). Conversely, GLI did not impair the mixed gastrocnemius of F+OVX rats. Therefore, in patients taking sulphonylureas, these results support the potential for impaired vascular KATP channel function to compromise muscle and therefore exercise tolerance. Such an effect, if present, would likely contribute to adverse cardiovascular events in premenopausal females more than males.

Chloride channel inhibition improves neuromuscular function under conditions mimicking neuromuscular disorders.

The skeletal muscle Cl- channels, the ClC-1 channels, stabilize the resting membrane potential and dampen muscle fibre excitability. This study explored whether ClC-1 inhibition can recover nerve-stimulated force in isolated muscle under conditions of compromised neuromuscular transmission akin to disorders of myasthenia gravis and Lambert-Eaton syndrome. Nerve-muscle preparations were isolated from rats. Preparations were exposed to pre-or post-synaptic inhibitors (ω-agatoxin, elevated extracellular Mg2+ , α-bungarotoxin or tubocurarine). The potential of ClC-1 inhibition (9-AC or reduced extracellular Cl- ) to recover nerve-stimulated force under these conditions was assessed. ClC-1 inhibition recovered force in both slow-twitch soleus and fast-twitch EDL muscles exposed to 0.2µmol/L tubocurarine or 3.5mmol/L Mg2+ . Similarly, ClC-1 inhibition recovered force in soleus muscles exposed to α-bungarotoxin or ω-agatoxin. Moreover, the concentrations of tubocurarine and Mg2+ required for reducing force to 50% rose from 0.14±0.02µmol/L and 4.2±0.2mmol/L in control muscles to 0.45±0.03µmol/L and 4.7±0.3mmol/L in muscles with 9-AC respectively (P<.05, paired T test). Inhibition of acetylcholinesterase (neostigmine) and inhibition of voltage-gated K+ channels (4-AP) relieve symptoms in myasthenia gravis and Lambert-Eaton syndrome, respectively. Neostigmine and 9-AC additively increased the tubocurarine concentration required to reduce nerve-stimulated force to 50% (0.56±0.05µmol/L with 9-AC and neostigmine) and, similarly, 4-AP and 9-AC additively increased the Mg2+ concentration required to reduce nerve-stimulated force to 50% (6.5±0.2mmol/L with 9-AC and 4-AP). This study shows that ClC-1 inhibition can improve neuromuscular function in pharmacological models of compromised neuromuscular transmission.

Skeletal muscle phenotype signaling with ex vivo endurance-type dynamic contractions in rat muscle.

Skeletal muscle phenotype may influence the response sensitivity of myocellular regulatory mechanisms to contractile activity. To examine this, we employed an ex vivo endurance-type dynamic contraction model to evaluate skeletal muscle phenotype-specific protein signaling responses in rat skeletal muscle. Preparations of slow-twitch soleus and fast-twitch extensor digitorum longus skeletal muscle from 4-wk-old female Wistar rats were exposed to an identical ex vivo dynamic endurance-type contraction paradigm consisting of 40 min of stretch-shortening contractions under simultaneous low-frequency electrostimulation delivered in an intermittent pattern. Phosphorylation of proteins involved in metabolic signaling and signaling for translation initiation was evaluated at 0, 1, and 4 h after stimulation by immunoblotting. For both muscle phenotypes, signaling related to metabolic events was upregulated immediately after stimulation, with concomitant absence of signaling for translation-initiation. Signaling for translation-initiation was then activated in both muscle phenotypes at 1-4 h after stimulation, coinciding with attenuated metabolic signaling. The recognizable pattern of signaling responses support how our ex vivo dynamic muscle contraction model can be utilized to infer a stretch-shortening contraction pattern resembling stretch-shortening contraction of in vivo endurance exercise. Moreover, using this model, we observed that some specific signaling proteins adhering to metabolic events or to translation-initiation exhibited phosphorylation changes in a phenotype-dependent manner, whereas other signaling proteins exhibited phenotype-independent changes. These findings may aid the interpretation of myocellular signaling outcomes adhering to mixed muscle samples collected during human experimental trials.NEW & NOTEWORTHY The application of cyclic ex vivo dynamic muscle contractions delivered in an intermittent pattern may be suitable for the exploration of skeletal muscle regulatory responses to endurance-type contractile activity. In the present study, it is demonstrated that the response to such stimulus of some nodal myocellular signaling proteins related to either metabolic or anabolic signaling events may be influenced by muscle phenotype, whereas the response of others appears to be independent of phenotype.

Foxm1 Modulates Cell Non-Autonomous Response in Zebrafish Skeletal Muscle Homeostasis.

foxm1 is a master regulator of the cell cycle, contributing to cell proliferation. Recent data have shown that this transcription factor also modulates gene networks associated with other cellular mechanisms, suggesting non-proliferative functions that remain largely unexplored. In this study, we used CRISPR/Cas9 to disrupt foxm1 in the zebrafish terminally differentiated fast-twitching muscle cells. foxm1 genomic disruption increased myofiber death and clearance. Interestingly, this contributed to non-autonomous satellite cell activation and proliferation. Moreover, we observed that Cas9 expression alone was strongly deleterious to muscle cells. Our report shows that foxm1 modulates a muscle non-autonomous response to myofiber death and highlights underreported toxicity to high expression of Cas9 in vivo.

Single cell RNA-seq analysis of the flexor digitorum brevis mouse myofibers

BackgroundSkeletal muscle myofibers can be separated into functionally distinct cell types that differ in gene and protein expression. Current single cell expression data is generally based upon single nucleus RNA, rather than whole myofiber material. We examined if a whole-cell flow sorting approach could be applied to perform single cell RNA-seq (scRNA-seq) in a single muscle type.MethodsWe performed deep, whole cell, scRNA-seq on intact and fragmented skeletal myofibers from the mouse fast-twitch flexor digitorum brevis muscle utilizing a flow-gated method of large cell isolation. We performed deep sequencing of 763 intact and fragmented myofibers.ResultsQuality control metrics across the different gates indicated only 171 of these cells were optimal, with a median read count of 239,252 and an average of 12,098 transcripts per cell. scRNA-seq identified three clusters of myofibers (a slow/fast 2A cluster and two fast 2X clusters). Comparison to a public skeletal nuclear RNA-seq dataset demonstrated a diversity in transcript abundance by method. RISH validated multiple genes across fast and slow twitch skeletal muscle types.ConclusionThis study introduces and validates a method to isolate intact skeletal muscle myofibers to generate deep expression patterns and expands the known repertoire of fiber-type-specific genes.

CORP: quantification of human skeletal muscle carnosine concentration by proton magnetic resonance spectroscopy.

Noninvasive techniques to quantify metabolites in skeletal muscle provide unique insight into human physiology and enable the translation of research into practice. Proton magnetic resonance spectroscopy (1H-MRS) permits the assessment of several abundant muscle metabolites in vivo, including carnosine, a dipeptide composed of the amino acids histidine and beta-alanine. Muscle carnosine loading, accomplished by chronic oral beta-alanine supplementation, improves muscle function and exercise capacity and has pathophysiological relevance in multiple diseases. Moreover, the marked difference in carnosine content between fast-twitch and slow-twitch muscle fibers has rendered carnosine an attractive candidate to estimate human muscle fiber type composition. However, the quantification of carnosine with 1H-MRS requires technical expertise to obtain accurate and reproducible data. In this review, we describe the technical and physiological factors that impact the detection, analysis, and quantification of carnosine in muscle with 1H-MRS. We discuss potential sources of error during the acquisition and preprocessing of the 1H-MRS spectra and present best practices to enable the accurate, reliable, and reproducible application of this technique.

In silico evidence of cardiac troponin non‐classical secretion: new insights on the pathobiology of myocardial injury

Background Cardiac troponin is one of the most important biomarkers in cardiology. Traditionally, it has represented a biochemical sign of necrosis, a characteristic of acute myocardial infarction (AMI). However, with the arrival of high-sensitivity detection assays, cardiac troponin is now detected in healthy people and in a number of uncommon scenarios unrelated to AMI (such as strenuous exercise, rapid atrial pacing and COVID-19). This suggest that troponin could be released as a consequence of alternative processes other than the well-stablished necrotic cell death. To try to explain this phenomenon, multiple mechanisms of troponin release have been postulated, although there is still no consensus on this matter. Furthermore, the term myocardial injury was included in the Fourth Universal Definition of Myocardial Infarction. This entity is understood as a troponin determination above the upper reference limit, not necessarily associated with a specific diagnosis. Nonetheless, the histological or pathobiological process responsible of myocardial injury has not been determined. Here, we studied the possibility of troponin release through non-classical secretion, a pathway specially triggered by cellular stress and inflammation. Methods The protein sequences of the eight different isoforms of troponin (I, T and C of fast-twitch skeletal, slow-twitch skeletal and cardiac muscle) were retrieved from the UniProtKB database. SecretomeP 2.0, a neural networks-based program, was used to predict if any of these proteins undergoes non-classical release. SignalP 5.0 and TMHMM 2.0 were also applied to rule out the possibility of classical (endoplasmic reticulum/Golgi dependent) and Type IV (transmembrane) non-classical secretion. Results and Discussion After analyzing the sequences, the cardiac T isoform was predicted to be non-classically secreted, as well as the I, T and C subunits of the fast-twitch skeletal muscle and the I subunit of the slow-twitch skeletal muscle. Additionally, none of the troponin isoforms was found to be subject to classical or Type IV secretion. The present study provides in silico evidence of cardiac troponin T release by means of non-classical secretion. We believe that this potential new mechanism could contribute to the debate around cardiac troponin high-sensitivity assays. Thus, it might establish a theoretical frame capable of explaining troponin elevations by causes other than myocyte necrosis. Conclusions Troponin T secretion may help in the pathobiological definition of myocardial injury. There are already some experimental studies showing the release of troponin through extracellular microvesicles (a type of non-classical secretion) in various cell lines. Nonetheless, more work is needed to fully understand this intriguing phenomenon in cardiomyocytes.

Myofibrillar (Network) Structures in Skeletal Muscles: An Evolutionarily Conserved Adaptation?

Skeletal muscles play a central role in converting biological fuel to functional force via the contraction of myofibrils. Until recently, myofibrils were characterized as individual cylindrical structures that run parallel to each other along the length of the muscle cell. However, using nanoscale 3D imaging, our group demonstrated that the contractile apparatus of vertebrate muscle cells is comprised of a unified myofibrillar matrix in which myofibrils frequently connect to each other through branching and merging events. While this work established myofibrillar connectivity as a fundamental component of all striated muscle cell types in vertebrate models, including mice and humans, it is unknown whether myofibrillar connectivity is evolutionarily conserved across invertebrate models commonly used to investigate muscle cell biology. Here, we use 3-dimensional focused-ion beam electron microscopy (FIB-SEM) to evaluate myofibrillar ultrastructure in different striated muscle cell types from Drosophila melanogaster. All procedures were approved by the National Heart, Lung, and Blood Institute Animal Care and Use Committee and performed in accordance with the guidelines described in the Animal Care and Welfare Act (7 USC 2142 § 13). 3D reconstruction of myofibrillar structures revealed that, similar to vertebrates, the myofibrils within direct flight (DF), jump, and leg muscles of Drosophila link together to form highly connected myofibrillar networks. Alternatively, we found that myofibrils in indirect flight (IF) muscles do not connect to each other as each successive sarcomere was connected in series to form isolated myofibrils. Quantitative assessment of DF, jump, and leg muscles revealed that the frequency of sarcomere branching differs by muscle type with leg muscles exhibiting the highest branching compared to no branching in IF muscles (see Supporting Figures). The percentage of myofibrils within the field of view of our datasets with at least one branching sarcomere is as follows: 94 ± 4.2% (mean ± SE, 3 cells, 150 myofibrils, 690 sarcomeres) in leg muscles, 77 ± 5.0% (4 cells, 200 myofibrils, 1461 sarcomeres) in DF muscles, 50 ± 3.7% (3 cells, 150 myofibrils, 1713 sarcomeres) in jump muscles, and 0 ± 0 % (3 cells, 114 myofibrils, 982 sarcomeres) in IF muscles. Classifying the analyzed muscles by specialized biological functions (slow oxidative: leg muscle; and fast oxidative/glycolytic: jump, DF, and IF muscles) suggests that branching events are more frequent in slow-twitch muscles compared to fast-twitch muscles. Since this frequency pattern arises in both vertebrates and invertebrates, we posit that sarcomere branching is an evolutionarily conserved adaptation. Therefore, the long-standing belief that sarcomeres are only arranged in a linear fashion (i.e., end to end in series) must be revisited to improve our understanding of how muscle architecture impacts biological processes and musculoskeletal pathologies.

Glycogen Synthase Kinase 3 activation status in soleus and EDL muscles from space‐flown mice aboard the BION‐M1 biosatellite

Background A significant limitation to long-term spaceflight and space habitation is the deterioration of human musculoskeletal health. It is well known that prolonged exposure to microgravity leads to pronounced muscle and bone loss, with consequences mimicking several major effects observed in human aging (i.e. osteoporosis and sarcopenia). It is therefore critical to elucidate the underlying molecular mechanisms of these processes to develop meaningful intervention strategies. Glycogen synthase kinase 3 (GSK3) is a serine/threonine protein kinase initially defined in its role of regulating glycogen homeostasis, though it's now known to interact with more than 100 target proteins. GSK3 has notably proven to negatively regulate muscle mass by inhibiting hypertrophy-related mechanisms, myoblast fusion, and muscle regeneration, while also promoting ubiquitin-mediated protein degradation and muscle inflammation. In this study, we explored GSK3 expression and its serine phosphorylation status, known to control its activation/inactivation, in soleus and extensor digitorum longus (EDL) muscles obtained from the BION-M1 mission. Methods Male C57BL6/J mice (3 months old) were flown in space for 1 month aboard the BION-M1 biosatellite (F) and were compared to a ground control (GC, space housing and diet conditions) and a flight control (FC, standard housing conditions) group. Soleus and EDL muscles were extracted and homogenized in protein extraction buffer. Western blotting was used to examine total and phosphorylated (i.e. inactivated) forms of GSK3 (ser21 GSK3α; ser9 GSK3β). Results In the soleus muscle, total GSK3α and GSK3β were reduced in the F group. Though we did not observe any changes in ser21 (GSK3α) or ser9 (GSK3β) phosphorylation, the ratio of phosphorylated:total GSK3β was significantly elevated in the F group compared with GC and FC. We did not observe any differences in soleus phosphorylated:total GSK3α. Further, we did not observe any differences in total, phosphorylated or phosphorylated:total GSK3α/β content in the EDL F group compared to GC and FC. Conclusions Altogether, our data suggest that in response to 1 month of spaceflight, GSK3 is less active in the soleus muscle through changes in expression and phosphorylation. GSK3 may therefore not be involved in the later stages of soleus atrophy in flown mice. This may be explained by a slow-to-fast fibre shift in soleus muscle, as GSK3 presence and activity is typically reduced in fast-twitch muscle fibres compared to slow-twitch. Future studies with soleus muscles from two other NASA-flown missions (RR1 and RR9) will be conducted to confirm these effects.

Role of Mitochondrial Calcium in the Maintenance of Skeletal Muscle Homeostasis

PV is a member of the EF-hand Ca2+-binding family of proteins, expressed in fast-twitch muscle fibers where, acting as a soluble cytoplasmic Ca2+ buffer, contributes to musclerelaxation by removing Ca2+ from the cytosol and facilitating its transport back to the SR. Furthermore, PV is one of the most downregulated “atrogenes”, the family of genes commonly up- and downregulated in both systemic and disuse-induced muscle atrophy. We exploited the murine strain lacking PV (PV-/-) to explore the link between PV function as Ca2+ buffer and as determinant of muscle trophism. First, we confirmed that PV expression is downregulated in denervated muscles and, surprisingly, we found a trend to a lower muscle atrophy in PV-/- than in wild type (WT) mice. Then, by acutely silencing and overexpressing PV in adult muscles of WT mice, we discovered that downregulation was accompanied by hypertrophy and upregulation by atrophy. The lack of PV had a minor impact on sarcoplasmic reticulum and affected the kinetics but not the amplitude of the cytosolic Ca2+ transients. In contrast, PV ablation was associated with an increased Ca2+ uptake in mitochondria, partly supported by MCU increased expression and accompanied by increased mitochondria size and number. Intriguingly, MCU silencing abolished the hypertrophic effect of PV ablation, suggesting a mitochondrial Ca2+-dependent mechanism of control of muscle trophism. This was corroborated by the enhanced expression of the mitochondrial Ca2+-dependent PGC-1α4 that promotes skeletal muscle hypertrophy. MCU silencing prevented increase of PGC-1αα4 expression and PGC-1α4 silencing prevented muscle fiber hypertrophy. These data reveal a novel role of PV that links the control of cytosolic Ca2+ concentration to mitochondrial adaptations, ultimately leading to muscle mass regulation. We believe that our results further underline that the mitochondrial pathway and its regulation are of utmost importance to uncover the molecular mechanisms of muscle atrophy.

The effect of denervation on myosin chaperone UNC45B in mice skeletal muscle

I. Background UNC45B, a myosin chaperone, affects myosin expression in C.elegans and has been reported to decrease with age in rats and humans. Therefore, it is thought that the decrease in UNC45B in the elderly may lead to lowering myosin level and the inability to regulate muscle quality, resulting in sarcopenia, an age-related loss of skeletal muscle mass. However, the mechanism by which UNC45B decreases with age remains unclear. In sarcopenia, atrophy occurs mainly in fast-twitch muscle fibers. Similarly, denervation leads to muscle atrophy mainly in fast-twitch muscle fibers, and therefore, it has been used as a model for sarcopenia. If UNC45B is reduced by muscle inactivity due to aging, it would be also reduced by inactivity due to denervation. Thus, we examined UNC45B expression in denervated muscle. II. Methods The right hindlimb muscles of 13-week-old male C57BL/6J mice were denervated by cutting and removing a 2 mm segment of the sciatic nerve, while the left hindlimb muscles served as sham-operated control. Two weeks after denervation, the gastrocnemius muscle was removed and weighed. Then the solution containing the homogenized muscle was centrifuged at 10000 g and the supernatant was used for Western blotting. Ⅲ. Results The muscle weight of the gastrocnemius muscle was decreased by about 50% and that myosin level was also significantly decreased by denervation (p<0.01), but the amount of UNC45B protein did not differ from that of the control leg. Although muscle protein synthesis measured by the SUnSET method was not changed by denervation, the amount of ubiquitinated protein was increased (p<0.001). Total and phosphorylation levels of Akt (an upstream regulator of the mechanistic target of rapamycin complex 1 (mTORC1) that facilitates protein synthesis), 4EBP1, and rpS6 (downstream targets of mTORC1) were increased by denervation (p<0.01). p62, LC3-I, and LC3-II, markers of autophagy, were increased in the denervated muscle (p62: p<0.05, LC3-I, LC3-II: p<0.01). Heat shock proteins that also act as chaperones for myosin, HSP90 and HSP73 were increased by denervation (HSP90: p<0.001, HSP73: p<0.05), while HSP70 was decreased (p<0.05). IV. Discussion Two weeks of denervation resulted in a significant gastrocnemius muscle weight decrease. However, no change had observed in UNC45B levels, indicating that UNC45B may not be involved in muscle atrophy caused by denervation, and the age-related decrease in UNC45B may also be not due to a decreased muscle activity. Denervation stimulated the protein degradation system while it did not affect muscle protein synthesis, suggesting that denervation-induced muscle atrophy may cause by enhancement of the degradation system. V. Conclusion In this study, there was no decrease in UNC45B after 2 weeks of denervation, suggesting that the decrease in UNC45B due to aging is not directly related to the decrease in muscle activity.

Fast skeletal myosin-binding protein-C regulates fast skeletal muscle contraction

Fast skeletal myosin-binding protein-C (fMyBP-C) is one of three MyBP-C paralogs and is predominantly expressed in fast skeletal muscle. Mutations in the gene that encodes fMyBP-C, MYBPC2, are associated with distal arthrogryposis, while loss of fMyBP-C protein is associated with diseased muscle. However, the functional and structural roles of fMyBP-C in skeletal muscle remain unclear. To address this gap, we generated a homozygous fMyBP-C knockout mouse (C2-/-) and characterized it both in vivo and in vitro compared to wild-type mice. Ablation of fMyBP-C was benign in terms of muscle weight, fiber type, cross-sectional area, and sarcomere ultrastructure. However, grip strength and plantar flexor muscle strength were significantly decreased in C2-/- mice. Peak isometric tetanic force and isotonic speed of contraction were significantly reduced in isolated extensor digitorum longus (EDL) from C2-/- mice. Small-angle X-ray diffraction of C2-/- EDL muscle showed significantly increased equatorial intensity ratio during contraction, indicating a greater shift of myosin heads toward actin, while MLL4 layer line intensity was decreased at rest, indicating less ordered myosin heads. Interfilament lattice spacing increased significantly in C2-/- EDL muscle. Consistent with these findings, we observed a significant reduction of steady-state isometric force during Ca2+-activation, decreased myofilament calcium sensitivity, and sinusoidal stiffness in skinned EDL muscle fibers from C2-/- mice. Finally, C2-/- muscles displayed disruption of inflammatory and regenerative pathways, along with increased muscle damage upon mechanical overload. Together, our data suggest that fMyBP-C is essential for maximal speed and force of contraction, sarcomere integrity, and calcium sensitivity in fast-twitch muscle.

Parsing the microRNA genetics basis regulating skeletal muscle fiber types and meat quality traits in pigs.

Muscle fibers are closely related to human diseases and livestock meat quality. However, the genetics basis of microRNAs (miRNAs) in regulating muscle fibers is not completely understood. In this study, we constructed the whole genome-wide miRNA expression profiles of porcine fast-twitch muscle [biceps femoris (Bf)] and slow-twitch muscle [soleus (Sol)], and identified hundreds of miRNAs, including four skeletal muscle-highly expressed miRNAs, ssc-miR-378, ssc-let-7f, ssc-miR-26a, and ssc-miR-27b-3p. Moreover, we identified 63 differentially expressed (DE) miRNAs between biceps femoris vs. soleus, which are the key candidate miRNAs regulating the skeletal muscle fiber types. In addition, we found that the expression of DE ssc-miR-499-5p was significantly correlated to the expression of Myoglobin (r=0.6872, P<0.0001) and Myosin heavy chain 7 (MYH7; r=0.5408, P=0.0020), and pH45min (r=0.3806, P=0.0380) and glucose content (r=-0.4382, P=0.0154); while the expression of DE ssc-miR-499-3p was significantly correlated to the expression of Myoglobin (r=0.5340, P=0.0024) and pH45min (r=0.4857, P=0.0065). Taken together, our data established a sound foundation for further studies on the regulatory mechanisms of miRNAs in skeletal muscle fiber conversion and meat quality traits in livestock, and could provide a genetic explanation of the role of miRNAs in human muscular diseases.

LncRNA-FKBP1C regulates muscle fiber type switching by affecting the stability of MYH1B

Long non-coding RNAs (lncRNAs) are well-known to participate in a variety of important regulatory processes in myogenesis. In our previous RNA-seq study (accession number GSE58755), we found that lncRNA-FKBP1C was differentially expressed between White Recessive Rock (WRR) and Xinghua (XH) chicken. Here, we have further demonstrated that lncRNA-FKBP1C interacted directly with MYH1B by biotinylated RNA pull-down assay and RNA immunoprecipitation (RIP). Protein stability and degradation experiments identified that lncRNA-FKBP1C enhanced the protein stability of MYH1B. Overexpression of lncRNA-FKBP1C inhibited myoblasts proliferation, promoted myoblasts differentiation, and participated in the formation of skeletal muscle fibers. LncRNA-FKBP1C could downregulate the fast muscle genes and upregulate slow muscle genes. Conversely, its interference promoted cell proliferation, repressed cell differentiation, and drove the transformation of slow-twitch muscle fibers to fast-twitch muscle fibers. Similar results were observed after knockdown of the MYH1B gene, but the difference was that the MYH1B gene had no effects on fast muscle fibers. In short, these data demonstrate that lncRNA-FKBP1C could bound with MYH1B and enhance its protein stability, thus affecting proliferation, differentiation of myoblasts and conversion of skeletal muscle fiber types.

Effects of metabolizable protein concentration, amino acid profile, and fiber source on the messenger RNA expression of skeletal muscle in peripartum dairy cows

After parturition, dairy cows mobilize AA from skeletal muscle to meet metabolizable protein (MP) requirements. High mobilization may compromise cow health and longer-term milk production. Postpartum diets with higher MP concentrations, improved AA profiles, or MP increased at the expense of forages rather than nonforage fiber sources may attenuate muscle catabolism; however, the molecular mechanisms responsible need investigation. We evaluated mRNA expression in the longissimus dorsi of cows fed postpartum diets differing in MP concentration, AA profile, and fiber source. From 0 to 25 d after parturition, 40 multiparous cows received the following diets: (1) 13% deficient in MP (D-MP), (2) adequate in MP using primarily soy protein (A-MP), (3) adequate in MP using blends of proteins and individual AA to improve the AA profile (Blend), or (4) similar to Blend except additional protein replaced forage (Blend-fNDF). Biopsies were taken approximately -5, 7, and 25 d relative to parturition. Greater dietary MP concentration (D-MP vs. A-MP and Blend) decreased expression of genes related to protein synthesis (MTOR, RPS6KB1) and degradation (FOXO1), inflammation (IFNG, TLR4), and endoplasmic reticulum (ER) stress (HSPA5, DDIT) and increased genes associated with lipogenesis (PPARG) and glucose oxidation (LDH, MB). In Blend versus A-MP (i.e., effect of AA profile), expression related to apoptosis (CASP8) and inflammation (TNFA) decreased and genes associated with cell cycle progression (E2F1) and fast-twitch glycolytic muscle fiber type (MYH4) increased. Less forage (Blend-fNDF vs. Blend) decreased genes associated with lipogenesis (PPARG, ACACA) and ER stress (BCL2, DDIT3, EIF2AK3, PPP1R15A) and increased genes associated with inflammation (TNF), inhibition of myogenesis (MSTN), and autophagy (PEBP1). In summary and based on mRNA expression, increasing MP supply may attenuate muscle turnover and ER stress. However, an unbalanced AA supply reduced cell cycle progression and protein synthesis. Lower energy supplies may reduce cell growth and cause autophagy.

µ-Crystallin: A thyroid hormone binding protein.

μ-Crystallin is a NADPH-regulated thyroid hormone binding protein encoded by the CRYM gene in humans. It is primarily expressed in the brain, muscle, prostate, and kidney, where it binds thyroid hormones, which regulate metabolism and thermogenesis. It also acts as a ketimine reductase in the lysine degradation pathway when it is not bound to thyroid hormone. Mutations in CRYM can result in non-syndromic deafness, while its aberrant expression, predominantly in the brain but also in other tissues, has been associated with psychiatric, neuromuscular, and inflammatory diseases. CRYM expression is highly variable in human skeletal muscle, with 15% of individuals expressing ≥13 fold more CRYM mRNA than the median level. Ablation of the Crym gene in murine models results in the hypertrophy of fast twitch muscle fibers and an increase in fat mass of mice fed a high fat diet. Overexpression of Crym in mice causes a shift in energy utilization away from glycolysis towards an increase in the catabolism of fat via β-oxidation, with commensurate changes of metabolically involved transcripts and proteins. The history, attributes, functions, and diseases associated with CRYM, an important modulator of metabolism, are reviewed.

Loss of splicing factor IK impairs normal skeletal muscle development

BackgroundIK is a splicing factor that promotes spliceosome activation and contributes to pre-mRNA splicing. Although the molecular mechanism of IK has been previously reported in vitro, the physiological role of IK has not been fully understood in any animal model. Here, we generate an ik knock-out (KO) zebrafish using the CRISPR/Cas9 system to investigate the physiological roles of IK in vivo.ResultsThe ik KO embryos display severe pleiotropic phenotypes, implying an essential role of IK in embryonic development in vertebrates. RNA-seq analysis reveals downregulation of genes involved in skeletal muscle differentiation in ik KO embryos, and there exist genes having improper pre-mRNA splicing among downregulated genes. The ik KO embryos display impaired neuromuscular junction (NMJ) and fast-twitch muscle development. Depletion of ik reduces myod1 expression and upregulates pax7a, preventing normal fast muscle development in a non-cell-autonomous manner. Moreover, when differentiation is induced in IK-depleted C2C12 myoblasts, myoblasts show a reduced ability to form myotubes. However, inhibition of IK does not influence either muscle cell proliferation or apoptosis in zebrafish and C2C12 cells.ConclusionThis study provides that the splicing factor IK contributes to normal skeletal muscle development in vivo and myogenic differentiation in vitro.

Pathological mechanisms of vacuolar aggregate myopathy arising from a Casq1 mutation.

Mice with a mutation (D244G, DG) in calsequestrin 1 (CASQ1), analogous to a human mutation in CASQ1 associated with a delayed onset human myopathy (vacuolar aggregate myopathy), display a progressive myopathy characterized by decreased activity, decreased ability of fast twitch muscles to generate force and low body weight after one year of age. The DG mutation causes CASQ1 to partially dissociate from the junctional sarcoplasmic reticulum (SR) and accumulate in the endoplasmic reticulum (ER). Decreased junctional CASQ1 reduces SR Ca2+ release. Muscles from older DG mice display ER stress, ER expansion, increased mTOR signaling, inadequate clearance of aggregated proteins by the proteasomes, and elevation of protein aggregates and lysosomes. This study suggests that the myopathy associated with the D244G mutation in CASQ1 is driven by CASQ1 mislocalization, reduced SR Ca2+ release, CASQ1 misfolding/aggregation and ER stress. The subsequent maladaptive increase in protein synthesis and decreased protein aggregate clearance are likely to contribute to disease progression.