Fast Myosin Heavy Chain Isoforms Research Articles

Birth trauma simulated by vaginal distention induced possible irreversible changes in the composition of the fiber types in the external urethral sphincter of rats.

The external urethral sphincter (EUS) is crucial in urinary continence development. Understanding the morphological features of the EUS in female rats after vaginal distention (VD), using a model of birth trauma, would aid in evaluating its functional and metabolic properties. Our recent study demonstrated that the EUS in female rats expresses one slow (type 1) and two fast (types 2A and 2B) myosin heavy chain (MHC) isoforms. Our preliminary experiment revealed that type 2B isoform expression was markedly reduced in the EUS 4 weeks after VD. Here, we aimed to examine the expression patterns of these three types of MHC isoforms, and an embryonic MHC, a marker of regeneration fibers, in the EUS of rats 3 days and 1, 2, and 8 weeks after VD using immunofluorescence staining. Hence, type 2B fibers were selectively damaged early in post-VD and did not recover fully later. Muscle regeneration in the sphincter peaked 1 week after trauma using a marker of immature fibers, embryonic myosin heavy chain. Electron microscopy revealed that the EUS of female rats was composed of mitochondria-rich muscle fibers. Myoblasts or immature muscle fibers were discovered in the sphincter layer 1 week after trauma. These results suggest that myogenesis after VD may not contribute to restoring normal fiber composition in a female rat's EUS.

Bridging the muscle genome to phenome across multiple biological scales.

Muscle is highly hierarchically organized, with functions shaped by genetically controlled expression of protein ensembles with different isoform profiles at the sarcomere scale. However, it remains unclear how isoform profiles shape whole-muscle performance. We compared two mouse hindlimb muscles, the slow, relatively parallel-fibered soleus and the faster, more pennate-fibered tibialis anterior (TA), across scales: from gene regulation, isoform expression and translation speed, to force-length-velocity-power for intact muscles. Expression of myosin heavy-chain (MHC) isoforms directly corresponded with contraction velocity. The fast-twitch TA with fast MHC isoforms had faster unloaded velocities (actin sliding velocity, Vactin; peak fiber velocity, Vmax) than the slow-twitch soleus. For the soleus, Vactin was biased towards Vactin for purely slow MHC I, despite this muscle's even fast and slow MHC isoform composition. Our multi-scale results clearly identified a consistent and significant dampening in fiber shortening velocities for both muscles, underscoring an indirect correlation between Vactin and fiber Vmax that may be influenced by differences in fiber architecture, along with internal loading due to both passive and active effects. These influences correlate with the increased peak force and power in the slightly more pennate TA, leading to a broader length range of near-optimal force production. Conversely, a greater force-velocity curvature in the near-parallel fibered soleus highlights the fine-tuning by molecular-scale influences including myosin heavy and light chain expression along with whole-muscle characteristics. Our results demonstrate that the individual gene, protein and whole-fiber characteristics do not directly reflect overall muscle performance but that intricate fine-tuning across scales shapes specialized muscle function.

Myosin Heavy Chain Isoform Mrna Expression In Low- And High-capacity Running Rats

Differential expression of Myosin Heavy Chain (MHC) isoforms in skeletal muscle determines its metabolic and functional characteristics. Endurance exercise training is associated with fast-to-slow MHC isoform transition, and which is considered to mediate the beneficial effects of exercise on metabolic health. Rats genetically bred for low-capacity running (LCR) and high-capacity running (HCR) are characterized by the health benefits typically described after endurance exercise training. LCR and HCR rats differed in maximal running capacity 14 fold. PURPOSE: To quantify mRNA expression for slow (MHC-I) and fast (MCH-IIa, MCH-IIb) MHC isoforms in soleus, gastrocnemius and vastus skeletal muscle of LCR and HCR male adult rats, and determine if HCR rats have higher mRNA expression for slow, and lower mRNA expression for fast, MHC isoforms. METHODS: Muscle tissue from 5 HCR and 5 LCR rats was homogenized and mRNA was isolated. The mRNA content of MHC isoforms in muscle was quantified using reverse transcriptase polymerase chain reaction (RT-PCR), and standardized to the 18S mRNA content in muscle. Differences between groups were calculated using the comparative CT method (2-ΔΔCT). LCR were the control group. RESULTS: Expression of mRNA of the MHC isoforms differed between groups and the type of skeletal muscle analyzed. MHC-I mRNA expression was lower in the HCR compared to LCR across all muscle groups, but this difference (2-ΔΔCT) was more pronounced for the gastrocnemius muscle [LCR: 1.0 ± 0.6 (mean±SE); HCR: 0.3 ± 0.2; P = 0.08]. Although MHC-IIa mRNA expression appeared lower in soleus and vastus in the HCR, it was higher in the HCR compared to LCR in the gastrocnemius muscle [LCR: 1.0 ± 1.0; HCR: 4.6 ± 3.6; P = 0.07]. MHC-IIb mRNA expression was lower in the HCR compared to LCR across all muscle groups, but this difference was more pronounced for the soleus muscle [LCR: 1.0 ± 1.1; HCR: 0.2 ± 0.4; P = 0.08]. CONCLUSION: Genetic differences linked to the MHC transcriptome (i.e., decrease in MHC-I mRNA) in muscle do not explain the exercise performance observed in HCR rats. Changes in the muscle MHC transcriptome may not directly be responsible for the health benefits associated with exercise training. It remains to be determined on whether the differences we observed at the MHC mRNA level are translated to the MHC proteome level.

Evidence for Progressive Shifts Towards α‐Myosin Isoform with Paradoxical Increases in Cardiac Hemodynamic Loads in Post‐Natal Healthy Rats

IntroductionThe balance of fast (α) and slow (β) cardiac myosin heavy chain (MYHC) isoforms is known to change with both aging and disease. While pathological states with increased hemodynamic loads are often accompanied by shifts toward energy‐sparing β isoforms, the underlying mechanism(s) of these changes remain poorly understood. Neonatal rats are a promising model to study such cardiac MYHC remodeling, as they show a well‐documented transition (β to α) during post‐natal development. In these experiments, MYHC‐isoform switches and in vivo cardiac structure/hemodynamic changes were evaluated during post‐natal development.MethodsCardiac echocardiographic and systemic/left ventricular (LV) hemodynamics were evaluated in Sprague‐Dawley rats (n = 61) ranging from post‐natal day (PND) 3 to adulthood. Hemodynamics were obtained before/after challenges with sodium nitroprusside (SNP, 25 μg/kg IV) and/or phenylephrine (PE, 10 μg/kg IV) to study cardiovascular responsiveness. LV’s were also collected (n = 48) for myosin isoform analysis via SDS‐PAGE MYHC separating gels.ResultsAs expected, post‐natal rats showed a progressive decline in β‐MYHC expression from PND3 (1.29 ± 0.05 α:β) to adulthood when α‐MYHC was dominant (12.78 ± 2.51 α:β). Concomitantly, postnatal rats showed progressive increases (P<0.05) in systemic/LV pressures (MAP: 46 ± 1 at <PND14 to 103 ± 2 mmHg, P<0.05), LV mass (0.16 ± 0.02g at <PND14 to 0.84 ± 017g) and diastolic dimensions (79 ± 6 at <PND14 to 288 ± 14 μL), with vastly preserved heart rates. Pressor responses to PE increased during maturation, while all age groups were sensitive to SNP‐mediated vaso‐relaxation. Ejection fraction was preserved during postnatal maturation with young pups showing lower rates of LV pressure development (dPdtmax 4052 ± 125 at <PND14 vs. 9478 ± 231 mmHg/s, P<0.05) consistent with the early expression of β‐MYHC.ConclusionsTaken together, these data provide evidence of a maturing cardiovascular system in post‐natal rats showing progressive increases in systemic/ventricular pressures and cardiac dimensions, with paradoxical shifts towards α‐MYHC isoforms. In short, in rats, the cardiac β‐MYHC isoform consistently declines over maturation despite sustained increases in systemic load.

Expression of ALS-linked SOD1 Mutation in Motoneurons or Myotubes Induces Differential Effects on Neuromuscular Function In vitro

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that selectively affects upper and lower motoneurons. Dismantlement of the neuromuscular junction (NMJ) is an early pathological hallmark of the disease whose cellular origin remains still debated. We developed an in vitro NMJ model to investigate the differential contribution of motoneurons and muscle cells expressing ALS-causing mutation in the superoxide dismutase 1 (SOD1) to neuromuscular dysfunction. The primary co-culture system allows the formation of functional NMJs and fosters the expression of the ALS-sensitive fast fatigable type II-b myosin heavy chain (MHC) isoform. Expression of SOD1G93A in myotubes does not prevent the formation of a functional NMJ but leads to decreased contraction frequency and lowers the slow type I MHC isoform transcript levels. Expression of SOD1G93A in both motoneurons and myotubes or in motoneurons alone however alters the formation of a functional NMJ. Our results strongly suggest that motoneurons are a major factor involved in the process of NMJ dismantlement in an experimental model of ALS.

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.

3D analysis of capillary network in skeletal muscle of obese insulin-resistant mice.

In obesity, the skeletal muscle capillary network regresses and the insulin-mediated capillary recruitment is impaired. However, it has been shown that in the early stage of advanced obesity, an increased functional vascular response can partially compensate for other mechanisms of insulin resistance. The present study aimed to investigate the changes in the capillary network around individual muscle fibres during the early stage of obesity and insulin resistance in mice using 3D analysis. Capillaries and muscle fibres of the gluteus maximus muscles of seven high-fat-diet-induced obese and insulin-resistant mice and seven age-matched lean healthy mice were immunofluorescently labelled in thick transverse muscle sections. Stacks of images were acquired using confocal microscope. Capillary network characteristics were estimated by methods of quantitative image analysis. Muscle fibre typing was performed by histochemical analysis of myosin heavy chain isoforms on thin serial sections of skeletal muscle. Capillary length per muscle fibre length and capillary length per muscle fibre surface were increased by 27% and 23%, respectively, around small muscle fibres in obese mice, while there were no significant comparative differences around large fibres of obese and lean mice. Furthermore, the capillarization was larger around small compared to large fibres and there was a shift toward fast type myosin heavy chain isoforms, with no significant changes in muscle fibre diameters, tortuosity and anisotropy in obese mice. Overall, the results show that obese insulin-resistant mice have selective increase in capillarization around small predominantly intermediate muscle fibres, which is most likely related to the impaired glucose metabolism characteristic of type 2 diabetes.

ATP Citrate Lyase Regulates Myofiber Differentiation and Increases Regeneration by Altering Histone Acetylation

ATP citrate lyase (ACL) plays a key role in regulating mitochondrial function, as well as glucose and lipid metabolism in skeletal muscle. We report here that ACL silencing impairs myoblast and satellite cell (SC) differentiation, and it is accompanied by a decrease in fast myosin heavy chain isoforms and MYOD. Conversely, overexpression of ACL enhances MYOD levels and promotes myogenesis. Myogenesis is dependent on transcriptional but also other mechanisms. We show that ACL regulates the net amount of acetyl groups available, leading to alterations in acetylation of H3(K9/14) and H3(K27) at the MYOD locus, thus increasing MYOD expression. ACL overexpression in murine skeletal muscle leads to improved regeneration after cardiotoxin-mediated damage. Thus, our findings suggest a mechanism forregulating SC differentiation and enhancing regeneration, which might be exploited for devising therapeutic approaches for treating skeletal muscle disease.

Accentuated Eccentric Loading for Training and Performance: A Review.

Accentuated eccentric loading (AEL) prescribes eccentric load magnitude in excess of the concentric prescription using movements that require coupled eccentric and concentric actions, with minimal interruption to natural mechanics. This method has been theorized to potentiate concentric performance through higher eccentric loading and, thus, higher concentric force production. There is also evidence for favorable chronic adaptations, namely shifts to faster myosin heavy chain isoforms and changes in IIx-specific muscle cross-sectional area. However, research concerning the acute and chronic responses to AEL is inconclusive, likely due to inconsistencies in subjects, exercise selection, load prescription, and method of providing AEL. Therefore, the purpose of this review is to summarize: (1) the magnitudes and methods of AEL application; (2) the acute and chronic implications of AEL as a means to enhance force production; (3) the potential mechanisms by which AEL enhances acute and chronic performance; and (4) the limitations of current research and the potential for future study.

IGF1 stimulates greater muscle hypertrophy in the absence of myostatin in male mice.

Insulin-like growth factors (IGFs) and myostatin have opposing roles in regulating the growth and size of skeletal muscle, with IGF1 stimulating, and myostatin inhibiting, growth. However, it remains unclear whether these proteins have mutually dependent, or independent, roles. To clarify this issue, we crossed myostatin null (Mstn-/-) mice with mice overexpressing Igf1 in skeletal muscle (Igf1+) to generate six genotypes of male mice; wild type (Mstn+/+ ), Mstn+/-, Mstn-/-, Mstn+/+:Igf1+, Mstn+/-:Igf1+ and Mstn-/-:Igf1+ Overexpression of Igf1 increased the mass of mixed fibre type muscles (e.g. Quadriceps femoris) by 19% over Mstn+/+ , 33% over Mstn+/- and 49% over Mstn-/- (P < 0.001). By contrast, the mass of the gonadal fat pad was correspondingly reduced with the removal of Mstn and addition of Igf1 Myostatin regulated the number, while IGF1 regulated the size of myofibres, and the deletion of Mstn and Igf1+ independently increased the proportion of fast type IIB myosin heavy chain isoforms in T. anterior (up to 10% each, P < 0.001). The abundance of AKT and rpS6 was increased in muscles of Mstn-/-mice, while phosphorylation of AKTS473 was increased in Igf1+mice (Mstn+/+:Igf1+, Mstn+/-:Igf1+ and Mstn-/-:Igf1+). Our results demonstrate that a greater than additive effect is observed on the growth of skeletal muscle and in the reduction of body fat when myostatin is absent and IGF1 is in excess. Finally, we show that myostatin and IGF1 regulate skeletal muscle size, myofibre type and gonadal fat through distinct mechanisms that involve increasing the total abundance and phosphorylation status of AKT and rpS6.

Absolute quantification of myosin heavy chain isoforms by selected reaction monitoring can underscore skeletal muscle changes in a mouse model of amyotrophic lateral sclerosis.

Skeletal muscle fibers contain different isoforms of myosin heavy chain (MyHC) that define distinctive contractile properties. In light of the muscle capacity to adapt MyHC expression to pathophysiological conditions, a rapid and quantitative assessment of MyHC isoforms in small muscle tissue quantities would represent a valuable diagnostic tool for (neuro)muscular diseases. As past protocols did not meet these requirements, in the present study we applied a targeted proteomic approach based on selected reaction monitoring that allowed the absolute quantification of slow and fast MyHC isoforms in different mouse skeletal muscles with high reproducibility. This mass-spectrometry-based method was validated also in a pathological specimen, by comparison of the MyHC expression profiles in different muscles from healthy mice and a genetic mouse model of amyotrophic lateral sclerosis (ALS) expressing the SOD1(G93A) mutant. This analysis showed that terminally ill ALS mice have a fast-to-slow shift in the fiber type composition of the tibialis anterior and gastrocnemius muscles, as previously reported. These results will likely open the way to accurate and rapid diagnoses of human (neuro)muscular diseases by the proposed method. Graphical Abstract Methods for myosin heavy chain (MyHC) quantification: a comparison of classical methods and selected reaction monitoring (SRM)-based mass spectrometry approaches.

Temporal embryonic transcription of chicken fast skeletal myosin heavy chain isoforms in the single comb white leghorn

There are numerous factors that can significantly influence embryonic development in poultry and thus make simple days of incubation (chronological age) a less than perfect metric for studying embryonic physiology. The developmental fast skeletal muscle myosin (MyHC), the predominant protein in the Pectoralis major (PM), is temporally expressed as a cadre of highly specific developmental isoforms. In the study described herein, a novel molecular technology (NanoString) was used to characterize the myosin isoform transcriptional patterns in the PM of Single Comb White Leghorn (SCWL) embryos. NanoString technology is based on quantitative analysis of the transcriptome through digital detection and quantification of target mRNA transcripts. Total RNA was isolated and gene transcription quantified using NanoString in embryonic muscle samples collected daily from 6 through 19 days of incubation. Data were analyzed using the LOESS smoothing function at a 95% confidence level. The temporal transcription of MyHC isoforms obtained in this study was consistent with the literature at higher specificity and resolution, thus validating NanoString for use in gene transcription analyses. The results support a hypothesis that the transcription patterns of the embryonic MyHC isoforms may be used as molecular clocks to further investigate the developmental relationships underlying embryonic fast skeletal muscle growth and development.

Abnormally Small Neuromuscular Junctions in the Extraocular Muscles From Subjects With Idiopathic Nystagmus and Nystagmus Associated With Albinism.

PurposeInfantile nystagmus syndrome (INS) is often associated with abnormalities of axonal outgrowth and connectivity. To determine if this manifests in extraocular muscle innervation, specimens from children with idiopathic INS or INS and albinism were examined and compared to normal age-matched control extraocular muscles.MethodsExtraocular muscles removed during normal surgery on children with idiopathic INS or INS and albinism were immunostained for neuromuscular junctions, myofiber type, the immature form of the acetylcholine receptor, and brain-derived neurotrophic factor (BDNF) and compared to age-matched controls.ResultsMuscles from both the idiopathic INS and INS and albinism groups had neuromuscular junctions that were 35% to 71% smaller based on myofiber area and myofiber perimeter than found in age-matched controls, and this was seen on both fast and slow myosin heavy chain isoform–expressing myofibers (all P < 0.015). Muscles from subjects with INS and albinism showed a 7-fold increase in neuromuscular junction numbers on fast myofibers expressing the immature gamma subunit of the acetylcholine receptor. The extraocular muscles from both INS subgroups showed a significant increase in the number and size of slow myofibers compared to age-matched controls. Brain-derived neurotrophic factor was expressed in control muscle but was virtually absent in the INS muscles.ConclusionsThese studies suggest that, relative to the final common pathway, INS is not the same between different patient etiologies. It should be possible to modulate these final common pathway abnormalities, via exogenous application of appropriate drugs, with the hope that this type of treatment may reduce the involuntary oscillatory movements in these children.

Estrogens maintain skeletal muscle and satellite cell functions.

Estrogens have crucial roles in an extensive range of physiological functions regulating cellular proliferation and differentiation, development, homeostasis, and metabolism. Therefore, prolonged estrogen insufficiency influences various types of tissues expressing estrogen receptors (ERs). Although ERs are expressed in skeletal muscle and its stem cells, called satellite cells, how prolonged estrogen insufficiency affects their function remains unclear. In this study, we investigated the effect of estrogen reduction on muscle in young ovariectomized (OVX) female mice. We found that reduced estrogens resulted in muscle atrophy in a time-dependent manner. Muscle force generation was reduced in OVX mice. Interestingly, prolonged estrogen insufficiency shifted fiber types toward faster myosin heavy chain isoforms. The number of satellite cells per isolated myofiber was unchanged, while satellite cell expansion, differentiation, and self-renewal were all markedly impaired in OVX mice. Indeed, muscle regeneration was significantly compromised in OVX mice. Taken together, our results demonstrate that estrogens are essential for comprehensively maintaining muscle function with its insufficiency affecting muscle strength and regeneration in young female mice.

Effects of intergenic single nucleotide polymorphisms in the fast myosin heavy chain cluster on muscle fiber characteristics and meat quality in Berkshire pigs

The heterogeneity of myosin heavy chain (MyHC) isoforms is closely related to muscle fiber characteristics, and meat quality in pigs. The fast MyHC gene cluster on porcine chromosome 12 contains MYH2, MYH1, and MYH4, which encode three fast MyHC isoforms expressed in adult skeletal muscle. Here, we identified four intergenic single nucleotide polymorphisms (SNPs) in the gene cluster. Of these, SNP1 and SNP2, which were located near MYH2, were genotyped in 199 Berkshire pigs. SNP1 were significantly associated with the total fiber number (P=0.046) and intramuscular fat contents (P=0.041), and SNP2 had significant effects on type I fiber number/area compositions (P=0.039 and P=0.041, respectively), water holding capacity (drip loss, P=0.045; cooking loss, P=0.001), and meat color (P=0.003). Taken together, our results suggest that the intergenic SNPs near MYH2 can affect muscle fiber formation and meat quality by modifying the regulatory elements of this gene cluster in pigs.

Prox1 transcription factor regulates satellite cell and skeletal muscle fiber phenotype and function

Prospero related homeobox 1 (Prox1) transcription factor is important for the development of several organs, including the heart, liver and lymphatic vessels. Prox1 gene polymorphisms have recently been related to insulin resistance. We show here that Prox1 is expressed in adult slow skeletal muscle fibers and satellite cells, both in rodents and in humans. Prox1 expression is increased during mouse C2C12 cell differentiation and in primary human myoblast differentiation into myotubes, and its silencing leads to failure in differentiation. Mechanistically, Prox1 activates calcineurin signaling both in vivo in skeletal muscles and in C2C12 cells. Overexpression of Prox1 in fast skeletal muscle induced the expression of slow muscle fiber-related myofibrillar and calcium-signaling genes, while deletion of Prox1 lead to the upregulation of the fast myosin heavy chain isoforms in slow soleus muscle. With lineage tracing, we showed that Prox1 expressing satellite cells proliferate and contribute to muscle regeneration after cardiotoxin-induced muscle injury. After acute running exercise, the expression of Prox1 was downregulated in mouse gastrocnemius muscle. We have identified Prox1 as an important transcription factor regulating mammalian skeletal muscle fiber type and satellite cell differentiation and phenotype. This provides novel information how skeletal muscle function is regulated, and is important in terms of understanding normal skeletal muscle physiology and muscle diseases.

Bronchial but not Tracheal Smooth Muscle is Hypercontractile in an Equine Model of Severe Asthma

Asthma is an inflammatory disease of the airways characterized by airway hyperresponsiveness, an exaggerated bronchoconstrictive response of airway smooth muscle (ASM) to stimuli. However, whether asthmatic ASM is mechanically different from non-asthmatic ASM is still unclear. Most studies so far have been performed on the trachealis muscle but it might not be representative of smooth muscle in the more peripheral airways. Thus, we addressed this question in an equine model of asthma (horse with heaves) and compared the trachealis (TSM) and peripheral bronchial (BSM) airway smooth muscle mechanics.Muscle strips from de-epithelialized TSM and BSM (less than 10 mm in diameter) were mounted horizontally in an experimental chamber between a length controller and a force transducer (Aurora Scientific Inc.). Maximal velocity of shortening (Vmax), methacholine dose response and stress were measured.We found no difference in Vmax between TSM from control horses and horses with heaves (0.124±0.01vs. 0.119±0.01; control vs. heaves; P=0.8), whereas the BSM exhibited a significantly higher Vmax (0.114±0.01 vs. 0.239±0.02; P=0.03). Western blot analysis showed a significant increase of the (+)insert fast smooth muscle myosin heavy chain (SMMHC) isoform in BSM of heaves-affected horses (0.78±0.04 vs. 1.0±0.05, P=0.0092), while no such difference was found in TSM (0.91±0.05 vs.0.97±0.08;P=0.54). The expression of calponin was significantly increased in TSM of heaves-affected horses (0.92±0.15 vs. 2.5±0.57, P=0.037; but this was not observed in BSM (0.37±0.14 vs. 0.49±0.18; P=0.62). There were no differences detected in the expression of MLCK, total SMMHC and transgelin. Peripheral airways may have a greater exposure to inflammatory cells than the trachealis which may explain their differences in mechanics. Further studies await to determine the mechanism underlying in observed effect.Supported by: CIHR(MOP102718), NIH(RO1HL103405), RFBR(12-04-0076)

Frequency dependence of power and its implications for contractile function of muscle fibers from the digital flexors of horses.

The digital flexors of horses must produce high force to support the body weight during running, and a need for these muscles to generate power is likely limited during locomotion over level ground. Measurements of power output from horse muscle fibers close to physiological temperatures, and when cyclic strain is imposed, will help to better understand the in vivo performance of the muscles as power absorbers and generators. Skinned fibers from the deep (DDF) and superficial (SDF) digital flexors, and the soleus (SOL) underwent sinusoidal oscillations in length over a range of frequencies (0.5–16 Hz) and strain amplitudes (0.01–0.06) under maximum activation (pCa 5) at 30°C. Results were analyzed using both workloop and Nyquist plot analyses to determine the ability of the fibers to absorb or generate power and the frequency dependence of those abilities. Power absorption was dominant at most cycling frequencies and strain amplitudes in fibers from all three muscles. However, small amounts of power were generated (0.002–0.05 Wkg−1) at 0.01 strain by all three muscles at relatively slow cycling frequencies: DDF (4–7 Hz), SDF (4–5 Hz) and SOL (0.5–1 Hz). Nyquist analysis, reflecting the influence of cross‐bridge kinetics on power generation, corroborated these results. The similar capacity for power generation by DDF and SDF versus lower for SOL, and the faster frequency at which this power was realized in DDF and SDF fibers, are largely explained by the fast myosin heavy chain isoform content in each muscle. Contractile function of DDF and SDF as power absorbers and generators, respectively, during locomotion may therefore be more dependent on their fiber architectural arrangement than on the physiological properties of their muscle fibers.

Effects of Eccentrically and Concentrically Biased Training on Mouse Muscle Phenotype

The molecular adaptations specifically induced by different muscle contraction types have only been partially elucidated. We previously demonstrated that eccentric contractions in human quadriceps elicited proteome modifications that suggest a muscle fiber typology adaptation. We address this question in a more systematic way by examining here the effects of different running modes on the mouse muscle proteome and the muscle fiber typology. Male adult mice (C57BL6) were randomly divided into downhill running (DHR) (quadricipital eccentrically biased contractions), uphill running (UHR) (quadricipital concentrically biased contractions), and untrained control (CONT) groups. Running groups performed five training sessions on an inclined treadmill for 75 to 135 min · d(-1), and the quadriceps muscles were dissected 96 h after the last session. Muscle protein extracts of DHR and UHR groups (n = 4/group) were subjected to a two-dimensional difference in gel electrophoresis (2D-DIGE) analysis coupled with mass spectrometry. The assessment of fiber type, size, and number was performed on the rectus femoris of the three groups (n = 6/group) using myosin heavy chain immunohistochemistry. In the proteomic analysis, eight spots identified as the fast myosin heavy chain isoforms exhibited a lower abundance in DHR compared with UHR (P < 0.05, t-test). In contrast, adenosine triphosphate (ATP) synthase subunit α and tubulin β were more expressed in DHR (P < 0.05). A significant higher proportion of Type I and IIa fibers was found for DHR compared with UHR or CONT groups (P < 0.05, one-way ANOVA). Our data suggest that the eccentrically biased contractions in mice induced specific adaptations in protein expression and muscle fiber composition, which may reflect a more oxidative muscle phenotype. The differences in stress placed on the muscle between both trainings may be responsible for some unique adaptations resulting from the eccentrically biased training.

Myosin Heavy Chain-2b Transcripts and Isoform Are Expressed in Human Laryngeal Muscles

Three fast myosin heavy chain (MyHC) isoforms, i.e. MyHC-2a, -2x and -2b, are expressed in skeletal muscles of smaller mammals. In contrast, only MyHC-2a and -2x have been revealed in humans so far. The expression of MyHC isoforms is known to be wider in the functionally more specialized laryngeal muscles. Though mRNA transcripts of the MyHC-2b gene were found to be expressed in certain human skeletal and laryngeal muscles, the corresponding isoform has not been demonstrated in these muscles. To our knowledge, we are the first to demonstrate not only the expression of MyHC-2b transcripts using an in situ hybridization technique but also the corresponding protein, i.e. the MyHC-2b isoform, in some human laryngeal muscles by immunohistochemistry but not by polyacrylamide gel electrophoresis. Using a set of antibodies specific to MyHC isoforms, we demonstrated that MyHC-2b was always co-expressed with the major MyHC isoforms, not only with the fast ones (MyHC-2a and -2x) but with the slow isoform (MyHC-1) as well.