Fast Isoforms Research Articles

Effects of age on human skeletal muscle: A systematic review and meta-analysis of myosin heavy chain isoform protein expression, fiber size and distribution.

Human studies examining the cellular mechanisms behind sarcopenia, or age-related loss of skeletal muscle mass and function, have produced inconsistent results. A systematic review and meta-analysis were performed to determine the aging effects on protein expression, size and distribution of fibers with various myosin heavy chain (MyHC) isoforms. Study eligibility included MyHC comparisons between young (18-49 years) and older (≥ 60 years) adults, with 27 studies identified. Relative protein expression was higher with age for the slow-contracting MyHC I fibers, with correspondingly lower fast-contracting MyHC II and IIA values. Fiber sizes were similar with age for MyHC I, while smaller for MyHC II and IIA. Fiber distributions were similar with age. When separated by sex, the few studies that examined females showed atrophy of MyHC II and IIA fibers with age, but no change in MyHC protein expression. Additional analyses by measurement technique, physical activity, and muscle biopsied provided important insights. In summary, age-related atrophy in fast-contracting fibers lead to more of the slow-contracting, lower force-producing isoform in older male muscles, which helps explain their age-related loss in whole muscle force, velocity, and power. Exercise or pharmacological interventions that shift MyHC expression towards faster isoforms and/or increase fast-contracting fiber size should decrease the prevalence of sarcopenia. Our findings also indicate that future studies need to include or focus solely on females, measure MyHC IIA and IIX isoforms separately, examine fiber type distribution, sample additional muscles to the vastus lateralis, and incorporate an objective measurement of physical activity.

Proteostasis of heat shock protein 90 in skeletal muscles of the long-tailed ground squirrel during hibernation

We investigated changes in the content of heat shock protein 90 in m. soleus (comprised of mainly fibers expressing the MyHC slow isoform I) and m. gastrocnemius (composed of mainly fibers expressing the MyHC fast isoforms II) of the long-tailed ground squirrel Urocitellus undulatus in different periods of the annual cycle: summer activity (seasonal control), hypothermia/torpor, winter (interbout) activity. The content of the protein in both muscles was found not to change throughout the entire hibernation period despite the development of atrophic changes, more pronounced in fast m. gastrocnemius. The role of HSP90 in maintaining the stability of giant sarcomeric titin protein molecules is discussed with reference to animal's entry into and exit from hypothermia, when the activity of calpain proteases increases due to the increased content of Ca2+ in the cytosol of muscle cells; and with respect to the torpor, when the activity of calpains is, most likely, not inhibited completely. During the interbout activity with an observed increased titin turnover in squirrel's striated muscles, a constant content of HSP90 appears to be required for the correct folding of newly synthesized titin molecules and their integration into sarcomeres, as well as for the removal of misfolded titin molecules and other proteins. Thus, HSP90 proteostasis in skeletal muscles of the long-tailed ground squirrel can contribute to maintaining a steady-state level of titin and, possibly, other sarcomeric proteins during hibernation, which, in turn, will contribute to maintaining a highly ordered sarcomeric structure and the necessary level of muscle contractile activity in different phases of the torpor-arousal cycle.

Shotgun proteomics characterization of potential allergens in dried and powdered krill and fresh and powdered whiteleg shrimp

A shotgun proteomic and in-silico analysis was performed to identify and characterize the proteomes and potential allergens in both processed and non-processed forms of krill (Acetes spp.) and whiteleg shrimp (Litopenaeusvannamei). The samples were analyzed using LC-MS/MS, and the identified proteins were analyzed for protein-protein interactions and Gene Ontology. Potential allergens were analyzed by Allermatch and predicted IgE epitope by AlgPred. The results revealed that processed krill and whiteleg shrimp had a higher number of identified proteins and potential allergens compared to their non-processed forms. The major functions of identified proteins were related to organelle organization, intracellular and cytoskeletal protein binding. Eleven potential allergens were identified in all samples, indicating common allergens in both species. In-silico analysis of potential allergens of krill and whiteleg shrimp showed IgE epitopes on alpha-actinin sarcomeric-like isoform X1, tropomyosin fast isoform and tropomyosin isoform X19. The peptide IQLLEEDLER in tropomyosin fast isoform was identified as a potential allergen marker in fresh, dried and powdered forms of krill and whiteleg shrimp. These findings provide insight into the food safety, clinical and diagnostic aspects of krill and shrimp, particularly regarding its potential allergenicity and its allergen biomarkers.

Acute fatigue induces paradoxical increases in velocity and power in a sex and myosin isoform dependent manner

OBJECTIVE or our study was to identify effects of acute fatigue on myofibrillar components and their function that persist beyond modifications to intracellular metabolic perturbations. Our HYPOTHESIS was that acute fatigue would induce differential phosphorylation in regulatory proteins in the sarcomere that would reduce tension and increase velocity and power. We used the following METHODS to test that hypothesis. Seven healthy, competitive athletes were recruited to participate (3 male, 4 female), entailing one session of unilateral fatiguing knee extension exercise to the point of task failure. Immediately after, bilateral percutaneous needle muscle biopsy was performed on the vastus lateralis muscle, providing a “fatigued” and “not fatigued” sample. Tissue was separated for phosphoproteomic analysis (western blot and isoelectric separation) and single fiber contractile mechanics (isotonic load clamps; isometric tension, velocity and power). Following mechanical assessment, myosin heavy chain (MHC) isoform was determined for each fiber via SDS-PAGE. Our RESULTS found no effect of fatigue on isometric tension across all fiber types. However, maximum shortening velocity was increased along with contractile power, consistent with our hypotheses. Surprisingly, our results varied by sex and fiber type, such that velocity was increased primarily in MHC IIA/X fibers in males, whereas females demonstrated a blunted response to fatigue. While baseline variation in regulatory protein phosphorylation (Myosin Binding Protein C; MyBP-C) were significantly correlated with power output in contractile power in fast isoforms (MHC IIA, MHC IIA/X) we did not detect significant alterations in phosphorylation of this protein with fatigue. We CONCLUDE that fatigue causes paradoxical increased in velocity and power that are particularly evident in males. While our measurement of MyBP-C phosphorylation did not detect effects of fatigue, other potential sarcomeric proteins may explain this paradoxical response to acute fatigue. Wu Tsai Human Performance Alliance This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Maintenance of AMPK Activity Eliminates Abnormally Accelerated Differentiation of Primary Myoblasts Isolated from Atrophied Rat Soleus Muscle

Mechanical unloading of skeletal muscles leads to the development of atrophic processes and a decrease in the total number of satellite cells (SCs) that are involved in muscle regeneration. In vitro studies revealed an increased differentiation of myoblasts derived from rat soleus muscle after an unloading-induced decrease in AMP-activated protein kinase (AMPK). AMPK is necessary for the activation of SCs and also participates in the regulation of myoblast proliferation and differentiation. It can be assumed that a decrease in the activity of AMPK after mechanical unloading can contribute to the acceleration of myoblast differentiation. The main purpose of this study was to elucidate a possible role of AMPK in the regulation of differentiation of myoblasts isolated from rat soleus muscle after mechanical unloading. To test this hypothesis, a specific AMPK activator, AICAR, was used to prevent a decrease in AMPK activity during differentiation of myoblasts isolated from rat soleus muscle after 7-day unloading. Immunocytochemistry, PCR-RT and Western blotting were used to assess changes during myoblast differentiation. In differentiating myoblasts derived from the unloaded soleus muscle there was a significant decrease in AMPK (Thr172) and ACC (Ser 79) phosphorylation levels, an increase in myotube differentiation index, myoblast fusion factors and the expression of myogenic regulatory factors (MRF). Furthermore, there was a decrease in the expression of slow myosin heavy chains (MyHC) and an increase in the expression of fast MyHC isoforms. AICAR treatment of differentiating myoblasts obtained from the unloaded soleus muscle prevented a decrease in AMPK and ACC phosphorylation, returned the expression levels of MRF and fast isoforms of MyHC to the control levels as well as maintained the expression of slow MyHC. Thus, abnormally accelerated differentiation of myoblasts isolated from atrophied rat soleus muscle can be compensated by maintaining the control levels of AMPK activity using AICAR.

Combined effects of heavy ion exposure and simulated Lunar gravity on skeletal muscle

BackgroundThe limitations to prolonged spaceflight include unloading-induced atrophy of the musculoskeletal system which may be enhanced by exposure to the space radiation environment. Previous results have concluded that partial gravity, comparable to the Lunar surface, may have detrimental effects on skeletal muscle. However, little is known if these outcomes are exacerbated by exposure to low-dose rate, high-energy radiation common to the space environment. Therefore, the present study sought to determine the impact of highly charge, high-energy (HZE) radiation on skeletal muscle when combined with partial weightbearing to simulate Lunar gravity. We hypothesized that partial unloading would compromise skeletal muscle and these effects would be exacerbated by radiation exposure. MethodsFor month old female BALB/cByJ mice were -assigned to one of 2 groups; either full weight bearing (Cage Controls, CC) or partial weight bearing equal to 1/6th bodyweight (G/6). Both groups were then divided to receive either a single whole body absorbed dose of 0.5 Gy of 300 MeV 28Si ions (RAD) or a sham treatment (SHAM). Radiation exposure experiments were performed at the NASA Space Radiation Laboratory (NSRL) located at Brookhaven National Laboratory on Day 0, followed by 21 d of CC or G/6 loading. Muscles of the hind limb were used to measure protein synthesis and other histological measures. ResultsTwenty-one days of Lunar gravity (G/6) resulted in lower soleus, plantaris, and gastrocnemius muscle mass. Radiation exposure did not further impact muscle mass. 28Si exposure in normal ambulatory animals (RAD+CC) did not impact gastrocnemius muscle mass when compared to SHAM+CC (p>0.05), but did affect the soleus, where mass was higher following radiation compared to SHAM (p<0.05). Mixed gastrocnemius muscle protein synthesis was lower in both unloading groups. Fiber type composition transitioned towards a faster isoform with partial unloading and was not further impacted by radiation. The combined effects of partial loading and radiation partially mitigated fiber cross-sectional area when compared to partial loading alone. Radiation and G/6 reduced the total number of myonuclei per fiber while leading to elevated BrdU content of skeletal muscle. Similarly, unloading and radiation resulted in higher collagen content of muscle when compared to controls, but the effects of combined exposure were not additive. ConclusionsThe results of this study confirm that partial weightbearing causes muscle atrophy, in part due to reductions of muscle protein synthesis in the soleus and gastrocnemius as well as reduced peripheral nuclei per fiber. Additionally, we present novel data illustrating 28Si exposure reduced nuclei in muscle fibers despite higher satellite cell fusion, but did not exacerbate muscle atrophy, CSA changes, or collagen content. In conclusion, both partial loading and HZE radiation can negatively impact muscle morphology.

Normal muscle fiber type distribution is recapitulated in aged ephrin-A3-/- mice that previously lacked most slow myofibers.

Individual limb muscles have characteristic representation and spatial distribution of muscle fiber types (one slow and up to three fast isoforms) appropriate to their unique anatomical location and function. This distribution can be altered by physiological stimuli such as training (i.e., for increased endurance or force) or pathological conditions such as aging. Our group previously showed that ephrin-A3 is expressed only on slow myofibers, and that adult mice lacking ephrin-A3 have dramatically reduced numbers of slow myofibers due to postnatal innervation of previously slow myofibers by fast motor neurons. In this study, fiber type composition of hindlimb muscles of aged and denervated/reinnervated C57BL/6 and ephrin-A3-/- mice was analyzed to determine whether the loss of slow myofibers persists across the lifespan. Surprisingly, fiber-type composition of ephrin-A3-/- mouse muscles at two years of age was nearly indistinguishable from age-matched C57BL/6 mice. After challenge with nerve crush, the percentage of IIa and I/IIa hybrid myofibers increased significantly in aged ephrin-A3-/- mice. While EphA8, the receptor for ephrin-A3, is present at all neuromuscular junctions (NMJs) on fast fibers in 3-6 mo old C57BL/6 and ephrin-A3-/- mice, this exclusive localization is lost with aging, with EphA8 expression now found on a subset of NMJs on some slow muscle fibers. This return to appropriate fiber-type distribution given time and under use reinforces the role of activity in determining fiber-type representation and suggests that, rather than being a passive baseline, the developmentally and evolutionarily selected fiber type pattern may instead be actively reinforced by daily living.

Differential expression of myosin heavy chain isoforms type II in skeletal muscles of polar and black bears.

In this study, the pattern of myosin heavy chain (MHC) isoforms expression in skeletal muscles of the trunk, forelimb and hindlimb in Polar Bear (PB) Ursus maritimus; American Black Bear (AmBB), Ursus americanus and Asian Black Bear (AsBB), Ursus thibetanus was analysed by immunohistochemistry and SDS-PAGE. Results showed that slow (MHC-I) and fast (MHC-II) isoforms exist in muscles of bears. Type II fibres were classified further into Type IIa and IIx in PB but not in AsBB and AmBB. The distribution of Type I and Type II fibres in the trunk, forelimb and hindlimb varied based on muscle type and animal species. The proportions of Type I fibres formed approximately one-third of muscle composition in PB (trunk, 32.0%; forelimb, 34.7%; hindlimb, 34.5%) and a half in both AsBB and AmBB whereas Type IIa and IIx formed approximately two-third in PB (trunk, 68.0%; forelimb, 65.3%; hindlimb, 65.5%) and a half of Type II in both AmBB and AsBB. PB is a good swimmer, lives in Arctic Ocean on slippery ice catching aquatic mammals such as seals and is larger in size compared to the medium sized AmBB (living in forest) and AsBB (arboreal). The results suggest that in bears, there is greater diversity in MHC isoforms II, being expressed in selected fast contracting skeletal muscles in response to variety of environments, weight bearing and locomotion.

Calretinin-Expressing Synapses Show Improved Synaptic Efficacy with Reduced Asynchronous Release during High-Rate Activity.

Calretinin (CR) is a major calcium binding protein widely expressed in the CNS. However, its synaptic function remains largely elusive. At the auditory synapse of the endbulb of Held, CR is selectively expressed in different subtypes. Combining electrophysiology with immunohistochemistry, we investigated the synaptic transmission at the endbulb of Held synapses with and without endogenous CR expression in mature CBA/CAJ mice of either sex. Two synapse subtypes showed similar basal synaptic transmission, except a larger quantal size in CR-expressing synapses. During high-rate stimulus trains, CR-expressing synapses showed improved synaptic efficacy with significantly less depression and lower asynchronous release, suggesting more efficient exocytosis than non-CR-expressing synapses. Conversely, CR-expressing synapses had a smaller readily releasable pool size, which was countered by higher release probability and faster synaptic recovery to support sustained release during high-rate activity. EGTA-AM treatment did not change the synaptic transmission of CR-expressing synapses, but reduced synaptic depression and decreased asynchronous release at non-CR-expressing synapses, suggesting that CR helps to minimize calcium accumulation during high-rate activity. Both synapses express parvalbumin, another calcium-binding protein with slower kinetics and higher affinity than CR, but not calbindin. Furthermore, CR-expressing synapses only express the fast isoform of vesicular glutamate transporter 1 (VGluT1), while most non-CR-expressing synapses express both VGluT1 and the slower VGluT2, which may underlie their lagged synaptic recovery. The findings suggest that, paired with associated synaptic machinery, differential CR expression regulates synaptic efficacy among different subtypes of auditory nerve synapses to accomplish distinctive physiological functions in transmitting auditory information at high rates.SIGNIFICANCE STATEMENT CR is a major calcium-binding protein in the brain. It remains unclear how endogenous CR impacts synaptic transmission. We investigated the question at the large endbulb of Held synapses with selective CR expression and found that CR-expressing and non-CR-expressing synapses had similar release properties under basal synaptic transmission. During high-rate activity, however, CR-expressing synapses showed improved synaptic efficacy with less depression, lower asynchronous release, and faster recovery. Furthermore, CR-expressing synapses use exclusive VGluT1 to refill synaptic vesicles, while non-CR-expressing synapses use both VGluT1 and the slower isoform of VGluT2. Our findings suggest that CR may play significant roles in promoting synaptic efficacy during high-rate activity, and selective CR expression can differentially impact signal processing among different synapses.

Definition of the mechanokinetic parameters accounting for the functional diversity of slow and fast isoforms of skeletal muscle myosin with a synthetic nanomachine

The mechanokinetic parameters characterising the force and power produced by fast (psoas) and slow (soleus) skeletal rabbit muscle are defined by feeding to a model simulation the mechanical output of a synthetic nanomachine (Pertici et al., Nat Commun 9:3532, 2018) powered by 8 heavy-mero-myosin fragments (HMM) of myosin II purified from either muscle. Implementation of a length clamp control for the minimization of the trap compliance allows to integrate the measurements of all the relevant steady state parameters (the isometric force (F0), the force-velocity relation and the maximum power (Pmax)) with transient kinetics parameters (rate of isometric force development (r) and force fluctuation due to individual motor attachment-detachment).

HDAC4 Is Indispensable for Reduced Slow Myosin Expression at the Early Stage of Hindlimb Unloading in Rat Soleus Muscle.

It is well known that reduced contractile activity of the main postural soleus muscle during long-term bedrest, immobilization, hindlimb unloading, and space flight leads to increased expression of fast isoforms and decreased expression of the slow isoform of myosin heavy chain (MyHC). The signaling cascade such as HDAC4/MEF2-D pathway is well-known to take part in regulating MyHC I gene expression. Earlier, we found a significant increase of HDAC4 in myonuclei due to AMPK dephosphorylation during 24 h of hindlimb unloading via hindlimb suspension (HU) and it had a significant impact on the expression of MyHC isoforms in rat soleus causing a decrease in MyHC I(β) pre-mRNA and mRNA expression as well as MyHC IIa mRNA expression. We hypothesized that dephosphorylated HDAC4 translocates into the nuclei and can lead to a reduced expression of slow MyHC. To test this hypothesis, Wistar rats were treated with HDAC4 inhibitor (Tasquinimod) for 7 days before HU as well as during 24 h of HU. We discovered that Tasquinimod treatment prevented a decrease in pre-mRNA expression of MyHC I. Furthermore, 24 h of hindlimb suspension resulted in HDAC4 nuclear accumulation of rat soleus but Tasquinimod pretreatment prevented this accumulation. The results of the study indicate that HDAC4 after 24 h of HU had a significant impact on the precursor MyHC I mRNA expression in rat soleus.

Heterozygous SOD2 deletion impairs SERCA function in soleus and heart muscles from female mice

Background Sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pumps actively transport intracellular Ca2+ back into the sarcoplasmic reticulum and are the primary facilitators of muscle relaxation, restoring intracellular [Ca2+] to resting levels. Like many proteins, SERCA pumps are subject to oxidation which may affect their functional capacity. Superoxide dismutase 2 (SOD2) is a critical antioxidant enzyme localised in the mitochondrial matrix which converts superoxide radicals into less reactive H2O2. The present study sought to explore the structural and functional consequences of halved SOD2 expression, particularly to SERCA isoforms, in skeletal and cardiac muscle. Specifically, we examined whether heterozygous SOD2 deletion would result in changes in levels of SERCA tyrosine nitration and S-glutathionylation. Tyrosine nitration is known to impair SERCA function, particularly SERCA2a (slow isoform) but not SERCA1a (fast isoform). Conversely, glutathionylation of SERCA may act as an adaptive modality to preserve function during moderate oxidative stress. Methods We obtained soleus, extensor digitorum longus (EDL), and left ventricle (LV) muscles from wild-type (WT) and SOD2 heterozygote (SOD2+/-) C57BL/6J female mice (n=8 per genotype, aged 6-7 months). Ca2+-dependent SERCA activity assays were performed along with immunoprecipitation experiments to examine SERCA2a and SERCA1a tyrosine nitration and S-glutathionylation. Results Heterozygous SOD2 deletion did not alter SERCA1a or SERCA2a expression in the soleus, EDL or LV compared with WT. However, relative to WT, SOD2+/- soleus muscles showed significant impairments in SERCA function, with a reduction in SERCA's affinity for Ca2+. This corresponded well with Western blot data showing significantly elevated SERCA2a tyrosine nitration and significantly reduced S-glutathionylation in the soleus. In contrast, we observed no change in SERCA function, SERCA1a tyrosine nitration, or S-glutathionylation in the EDL. Furthermore, there were no differences in SERCA1a tyrosine nitration in the soleus, which is consistent with the notion that the SERCA1a isoform is less susceptible to nitrosative modification. Together, these results suggest that the slow SERCA isoform is more susceptible to heterozygous SOD2 deletion, and this is further supported by a significant reduction in SERCA's affinity for Ca2+ in the LV where SERCA2a is the predominant isoform. Conclusions This is the first study showing the physiological effects of halved SOD2 expression on muscle SERCA function. Our results reinforce the idea that SERCA2a is more sensitive to nitrosative modification.

Increased Skeletal Muscle Fiber Cross-Sectional Area, Muscle Phenotype Shift, and Altered Insulin Signaling in Rat Hindlimb Muscles in a Prenatally Androgenized Rat Model for Polycystic Ovary Syndrome.

Women with polycystic ovary syndrome (PCOS) are reported to have greater lean mass and insulin resistance. To examine muscular changes in a prenatally androgenized (PNA) rat model for PCOS, Sprague–Dawley rats were exposed to 5 mg testosterone or vehicle daily on gestational days 16–19. At 15 weeks of age, endurance on a rota-rod treadmill was measured. At 16 weeks of age, fasting blood glucose and insulin, hindlimb skeletal muscle mass, muscle fiber cross-sectional area (CSA) and composition, and intra- and peri-muscular lipid droplets were examined. Expression of mitochondrial marker ATP synthase and insulin signaling proteins were also investigated. Compared with controls, PNA female rats demonstrated greater total body and hindlimb muscle weights, greater muscle fiber CSA, and trending reduced time on the rota-rod. An increase in fibers co-expressing the slow and fast isoforms of myosin (90 vs. 86%, p < 0.05) and greater expression of ATP synthase (6-fold, p < 0.005) were observed in the gastrocnemius (GN) muscle. More lipid content was observed in GN and tibialis anterior (TA) muscles. PNA rats had elevated fasting serum insulin (1.9 vs. 1.2 ng/mL, p < 0.005) but comparable fasting glucose. Expression of total and Ser636/9-phosphorylated IRS1 were altered in PNA rat hindlimb muscles. Together, skeletal muscle alterations in hindlimb muscles of a PNA rat model for PCOS may represent consequences of, or adaptations to, insulin resistance in this model.

Single-nucleus RNA-seq and FISH identify coordinated transcriptional activity in mammalian myofibers

Skeletal muscle fibers are large syncytia but it is currently unknown whether gene expression is coordinately regulated in their numerous nuclei. Here we show by snRNA-seq and snATAC-seq that slow, fast, myotendinous and neuromuscular junction myonuclei each have different transcriptional programs, associated with distinct chromatin states and combinations of transcription factors. In adult mice, identified myofiber types predominantly express either a slow or one of the three fast isoforms of Myosin heavy chain (MYH) proteins, while a small number of hybrid fibers can express more than one MYH. By snRNA-seq and FISH, we show that the majority of myonuclei within a myofiber are synchronized, coordinately expressing only one fast Myh isoform with a preferential panel of muscle-specific genes. Importantly, this coordination of expression occurs early during post-natal development and depends on innervation. These findings highlight a previously undefined mechanism of coordination of gene expression in a syncytium.

Innervation and electrical pulse stimulation - in vitro effects on human skeletal muscle cells.

Contraction-induced adaptations in skeletal muscles are well characterized in vivo, but the underlying cellular mechanisms are still not completely understood. Cultured human myotubes represent an essential model system for human skeletal muscle that can be modulated ex vivo, but they are quiescent and do not contract unless being stimulated. Stimulation can be achieved by innervation of human myotubes in vitro by co-culturing with embryonic rat spinal cord, or by replacing motor neuron activation by electrical pulse stimulation (EPS). Effects of these two in vitro approaches, innervation and EPS, were characterized with respects to the expression of myosin heavy chains (MyHCs) and metabolism of glucose and oleic acid in cultured human myotubes. Adherent human myotubes were either innervated with rat spinal cord segments or exposed to EPS. The expression pattern of MyHCs was assessed by quantitative polymerase chain reaction, immunoblotting, and immunofluorescence, while the metabolism of glucose and oleic acid were studied using radiolabelled substrates. Innervation and EPS promoted differentiation towards different fiber types in human myotubes. Expression of the slow MyHC-1 isoform was reduced in innervated myotubes, whereas it remained unaltered in EPS-treated cells. Expression of both fast isoforms (MyHC-2A and MyHC-2X) tended to decrease in EPS-treated cells. Both approaches induced a more oxidative phenotype, reflected in increased CO2 production from both glucose and oleic acid. Novelty: Innervation and EPS favour differentiation into different fiber types in human myotubes. Both innervation and EPS promote a metabolically more oxidative phenotype in human myotubes.

Single-molecule analysis reveals that regulatory light chains fine-tune skeletal myosin II function

Myosin II is the main force-generating motor during muscle contraction. Myosin II exists as different isoforms that are involved in diverse physiological functions. One outstanding question is whether the myosin heavy chain (MHC) isoforms alone account for these distinct physiological properties. Unique sets of essential and regulatory light chains (RLCs) are known to assemble with specific MHCs, raising the intriguing possibility that light chains contribute to specialized myosin functions. Here, we asked whether different RLCs contribute to this functional diversification. To this end, we generated chimeric motors by reconstituting the MHC fast isoform (MyHC-IId) and slow isoform (MHC-I) with different light-chain variants. As a result of the RLC swapping, actin filament sliding velocity increased by ∼10-fold for the slow myosin and decreased by >3-fold for the fast myosin. Results from ensemble molecule solution kinetics and single-molecule optical trapping measurements provided in-depth insights into altered chemo-mechanical properties of the myosin motors that affect the sliding speed. Notably, we found that the mechanical output of both slow and fast myosins is sensitive to the RLC isoform. We therefore propose that RLCs are crucial for fine-tuning the myosin function.

Dietary Effects on Fiber Type Plasticity in the Masseter Muscle

Clinical and experimental studies have shown the masticatory complex (the skeletal and soft tissues associated with feeding) to be phenotypically plastic with regard to dietary material properties. Harder, tougher diets generally produce larger skeletal elements with greater bone density. However, less is known about the plasticity of the chewing musculature. Muscles are composed of two primary fiber types: slow‐twitch (type I) and fast‐twitch (type II). Fast‐twitch fibers produce rapid, powerful contractions but fatigue more quickly than slow‐twitch fibers. Here we use a rodent model to examine the plasticity of the masseter muscle fibers in response to inter‐individual variation in dietary material properties. We hypothesize that rats raised on hard, tough diets will have more fast‐twitch muscle fibers with greater cross‐sectional area, representing an increase in force production by the masseter in response to the dietary material properties.Male Sprague‐Dawley rats were raised from weaning (3 weeks) to adulthood (16 weeks) in two dietary treatment groups: a “hard/tough” pellet diet and a “soft” meal diet. Post‐sacrifice, fixed muscles were embedded in paraffin and divided into anterior, middle, and posterior regions. To visualize fast‐twitch fibers, 6 sections per region per individual were stained with a primary antibody for the fast isoforms of myosin heavy chain (MHC) and a fluorescent secondary antibody. Images were collected at 4× using a fluorescent confocal microscope. ImageJ was used to collect total muscle cell area, fast‐twitch cell area, and slow‐twitch and fast‐twitch cell counts. Mann‐Whitney U tests were used to compare cell count and area between the treatment groups (n=5/group, α = 0.05).Preliminary results indicate that hard/tough diets tend to be associated with a decrease in slow‐twitch fiber count and an increase in fast twitch fiber cross‐sectional area in the rat masseter. This allows for a greater proportion of fast‐twitch muscle fibers in a given muscle volume. We propose that this change reflects an increase in muscle force production during feeding on mechanically challenging food items. The results of this study allow for greater understanding of skeletal muscle growth postnatally through adulthood, and provides insight into the consequences of material properties of diet for the function and dysfunction of the human masticatory complex. Future studies are needed to address how muscle fiber plasticity changes with age and with intra‐individual variation in the material properties of diet.Support or Funding InformationFunding was provided by the NSF (BCS‐1061368), the Wenner‐Gren Foundation, the American Society of Mammalogists, and the American Association for Anatomy Innovations Program.

In Situ Characterization of the Working Stroke of the Slow and Fast Isoforms of Muscle Myosin

Muscle performance depends on the myosin heavy chain (MHC) isoform expressed in the myosin II motor. Stiffness measurements at sarcomere level in skinned fibres from either soleus or psoas muscles of rabbit have recently indicated that the stiffness and the force of the slow myosin isoform (soleus), are three times smaller than those of the fast isoform (psoas) (Percario et al. J Physiol 596:1243, 2018). Here the investigation on the mechano-kinetic features of the two isoforms in situ is extended to the comparison of the size and speed of the working stroke, measured on the isotonic velocity transient elicited by stepwise drops in force of different sizes superimposed on the plateau force (T0) of maximally Ca2+ activated skinned fibres from either muscle (sarcomere length 2.4 μm, temperature 12°C, 4% dextran T-500). The analysis of the early rapid shortening, due to the synchronisation of the working stroke in the attached motors shows that at high load (0.8 T0) the size of the working stroke is smaller in the slow isoform (2 nm) than in the fast isoform (5 nm), while it is similar at low load (7-8 nm at 0.2 T0). The rate of the working stroke increases by ∼6 times with the decrease in load from 0.8 to 0.2 T0, but, at any load, is one order of magnitude slower in the slow isoform than in the fast isoform. A reduced size of the working stroke of the slow isoform, together with the smaller force per motor, opens the question on the mechanism that can explain the high efficiency of the slow muscle with respect to the fast muscle. Supported by MIUR-PRIN 2010R8JK2X and Telethon GGP12282 (Italy).

Evaluation of Myosin Heavy Chain Isoforms in Biopsied Longissimus Thoracis Muscle for Estimation of Meat Quality Traits in Live Pigs.

Simple SummaryPork quality has become an important parameter in the industry. Traditional pork quality was assessed postmortem. It is considered that the determination of meat quality in live pigs is beneficial in order to obtain better pork quality and to reduce cost in production. In the present study, myosin heavy chain (MHC) isoforms in both of the pre- and postmortem longissimus thoracis muscle were evaluated as novel parameters for meat quality estimation in pork by correlation and clustering analysis. MHC isoforms in live pigs could be applied in a practical and useful method to predict meat quality in pork.Estimating meat quality prior to slaughter will be beneficial for the rapid identification of specific traits or poor quality pork compared to a conventional assessment at postmortem. In this study, we identified and quantified myosin heavy chain (MHC) isoforms from a biopsied longissimus thoracis muscle of pigs, and determined their correlation with postmortem muscle fiber characteristics and meat quality. MHC slow and fast isoforms proportions from biopsied samples correlated with postmortem percentage of type I and type IIB muscle fibers, respectively (p < 0.05). The percentage of the biopsied MHC slow isoform showed a positive correlation with pH at 45 min postmortem, and negative correlations with filter-paper fluid uptake and drip loss in pork (p < 0.05). Furthermore, clustering the pigs into three groups based on the biopsied MHC isoform proportions was not only significantly associated with muscle fiber number and proportions of muscle fiber area, but also correlated with pH at 45 min postmortem and the National Pork Producers Council color score (p < 0.05). Collectively, our findings indicate that the biopsied MHC isoforms serve as parameter for estimating meat quality, with the association between the higher proportion of MHC slow isoforms and pH at 45 min postmortem in particular being indicative of better pork quality.

Ammonia Induces a Myostatin-Mediated Atrophy in Mammalian Myotubes, but Induces Hypertrophy in Avian Myotubes

Ammonia, a byproduct of protein catabolism that is generally regarded as toxic, is processed by the liver for excretion. In diseases resulting in hepatic insufficiency, circulating ammonia levels increase dramatically, ensuing secondary disorders. Sarcopenia, or loss of muscle mass, is commonly associated with hyperammonemia. In mammalian models of cirrhosis, increased myostatin is consistent, contributes to muscle autophagy, and reduces satellite cell activation and differentiation, whereas, avian species show a positive myogenic response to ammonia. The objective of the study was to elucidate the effect of ammonia in chicken, mouse, and rat derived myotubes. Primary myoblasts were isolated from the pectoralis major (breast) and biceps femoris (thigh) of embryonic day 17 chicken embryos, and from the hindlimbs of 3-day-old rat pups. C2C12 cells were used for mouse myoblasts. Myotubes were exposed to 10mM ammonium acetate (AA) or 10mM sodium acetate (SA) for 24 hours to determine myogenic response to ammonia. Relative expression of myostatin mRNA, determined by quantitative real-time PCR, was significantly higher in mammalian myotubes compared to chicken myotubes (P < 0.001). Western blot analysis of myostatin protein confirmed a significant increase in ammonia treated rat myotubes, while chicken breast myotubes showed a significant decrease in myostatin (P < 0.05). Myotube diameter significantly increased in chicken breast and thigh cultures treated with ammonia, while diameter was significantly reduced in mouse and rat myotubes (P < 0.05). Intracellular glutamine is significantly higher in chicken thigh, but not breast, myotubes treated with AA compared to SA treated myotubes (P < 0.05). To investigate fiber type differences in ammonia metabolism, Western blot analysis of protein from AA and SA treated myotubes was examined for fast and slow myosin heavy chain isoforms. AA treatment resulted in a higher ratio of fast to slow isoforms of myosin heavy chain in both types of chicken myotubes, while fast isoforms were decreased in AA treated mouse and rat myotubes. These data demonstrate that chicken myotubes respond positively to ammonia while rodent myotubes respond negatively. Further, there is evidence that ammonia induces a fast fiber type shift in avian muscle, but a slow phenotype shift in mammalian muscle.