Fast Myosin Research Articles

Abstract 17492: Chronic Treatment With a Mavacamten-Like Myosin-Modulator (MYK-581) Prevents Left-Atrial Remodeling, Decreases Cardiac Troponin Leakage, and Blunts Mortality in a Mini-Pig Model of Inherited Hypertrophic Cardiomyopathy

Introduction: Hypertrophic cardiomyopathy (HCM) is a progressive disease characterized by cardiac remodeling, impaired relaxation, left-atrial enlargement, and exertional intolerance. Direct myosin-inhibition with mavacamten can normalize contractility and improve exercise capacity in patients with obstructive HCM (oHCM). However, mavacamten also limits residual cross-bridges during diastole, and therefore, may offer cardiac benefits beyond obstruction relief. This in vivo study evaluated the chronic effects of MYK-581, a mavacamten surrogate, in a genetic large-animal model of HCM. Methods: Young cloned Yucatan mini-pigs with a heterozygous MYH7 R403Q mutation were randomly assigned to one of two arms: untreated-controls (CTRL, n = 29) or daily MYK-581 (n = 22; PO); wild-type pigs (WT) untreated served as disease-controls. After 14 weeks of treatment, pigs underwent in vivo cMR imaging, including T1 mapping and extracellular volume (ECV) assessments. In a subset of pigs, biomechanical studies were performed in skinned left-ventricular (LV) and left-atrial (LV) fibers. *, #: P<0.05 vs. CTRL or WT. Results: In HCM pigs, MYK-581 treatment decreased mortality (9.0* vs. 37.9% in CTRL). MYK-581 blunted cTnT leakage, reducing both absolute values (21.2 ± 3.2* vs. 34.0 ± 4.3 ng/L in CTRL) and the incidence of cTnT > 20 ng/L (36* vs. 81% in CTRL). Treated pigs had smaller LA volumes (16 ± 1* vs. 29 ± 4mL in CTRL) with lower LV T1-times and ECV (27 ± 1* vs. 32 ± 2% in CTRL). LA fibers from untreated HCM pigs showed biomechanical remodeling characteristic of chronic overload: increased maximal force (21.8 ± 1.5# vs. 14.7 ± 1.4 mN/mm2 in WT) and slowed cross-bridge formation rates (Ktr: 4.9 ± 0.4 # vs. 6.6 ± 0.6 s-1 in WT) consistent with a switch towards slow-myosin isoforms. MYK-581 prevented this remodeling (e.g., Ktr: 8.1 ± 1.3 s-1*), preserving normal LA (fast) myosin content. Conclusions: Chronic direct myosin attenuation with a mavacamten surrogate prevented left-atrial remodeling, a known prognostic indicator in HCM. Chronic treatment also reduced cardiac troponin leakage characteristic HCM, and, more importantly, decreased mortality. Taken together, these pre-clinical observations show potential salutary effects beyond obstruction relief in HCM.

ProNGF/p75NTR Axis Drives Fiber Type Specification by Inducing the Fast-Glycolytic Phenotype in Mouse Skeletal Muscle Cells.

Despite its undisputable role in the homeostatic regulation of the nervous system, the nerve growth factor (NGF) also governs the relevant cellular processes in other tissues and organs. In this study, we aimed at assessing the expression and the putative involvement of NGF signaling in skeletal muscle physiology. To reach this objective, we employed satellite cell-derived myoblasts as an in vitro culture model. In vivo experiments were performed on Tibialis anterior from wild-type mice and an mdx mouse model of Duchenne muscular dystrophy. Targets of interest were mainly assessed by means of morphological, Western blot and qRT-PCR analysis. The results show that proNGF is involved in myogenic differentiation. Importantly, the proNGF/p75NTR pathway orchestrates a slow-to-fast fiber type transition by counteracting the expression of slow myosin heavy chain and that of oxidative markers. Concurrently, proNGF/p75NTR activation facilitates the induction of fast myosin heavy chain and of fast/glycolytic markers. Furthermore, we also provided evidence that the oxidative metabolism is impaired in mdx mice, and that these alterations are paralleled by a prominent buildup of proNGF and p75NTR. These findings underline that the proNGF/p75NTR pathway may play a crucial role in fiber type determination and suggest its prospective modulation as an innovative therapeutic approach to counteract muscle disorders.

Alternative N-terminal regions of Drosophila myosin heavy chain II regulate communication of the purine binding loop with the essential light chain

We investigated the biochemical and biophysical properties of one of the four alternative exon-encoded regions within the Drosophila myosin catalytic domain. This region is encoded by alternative exons 3a and 3b and includes part of the N-terminal β-barrel. Chimeric myosin constructs (IFI-3a and EMB-3b) were generated by exchanging the exon 3-encoded areas between native slow embryonic body wall (EMB) and fast indirect flight muscle myosin isoforms (IFI). We found that this exchange alters the kinetic properties of the myosin S1 head. The ADP release rate (k-D ) in the absence of actin is completely reversed for each chimera compared with the native isoforms. Steady-state data also suggest a reciprocal shift, with basal and actin-activated ATPase activity of IFI-3a showing reduced values compared with wild-type (WT) IFI, whereas for EMB-3b these values are increased compared with wild-type (WT) EMB. In the presence of actin, ADP affinity (KAD ) is unchanged for IFI-3a, compared with IFI, but ADP affinity for EMB-3b is increased, compared with EMB, and shifted toward IFI values. ATP-induced dissociation of acto-S1 (K1k+2 ) is reduced for both exon 3 chimeras. Homology modeling, combined with a recently reported crystal structure for Drosophila EMB, indicates that the exon 3-encoded region in the myosin head is part of the communication pathway between the nucleotide binding pocket (purine binding loop) and the essential light chain, emphasizing an important role for this variable N-terminal domain in regulating actomyosin crossbridge kinetics, in particular with respect to the force-sensing properties of myosin isoforms.

Acto-Myosin Cross-Bridge Stiffness Depends on the Nucleotide State of Myosin II.

How various myosin isoforms fulfill the diverse physiological requirements of distinct muscle types remain unclear. Myosin II isoforms expressed in skeletal muscles determine the mechanical performance of the specific muscles. Here, we employed a single-molecule optical trapping method and compared the chemomechanical properties of slow and fast muscle myosin II isoforms. Stiffness of the myosin motor is key to its force-generating ability during muscle contraction. We found that acto-myosin (AM) cross-bridge stiffness depends on its nucleotide state as the myosin progresses through the ATPase cycle. The strong actin bound "AM.ADP" state exhibited >2 fold lower stiffness than "AM rigor" state. The two myosin isoforms displayed similar "rigor" stiffness. We conclude that the time-averaged stiffness of the slow myosin is lower due to prolonged duration of the AM.ADP state, which determines the force-generating potential and contraction speed of the muscle, elucidating the basis for functional diversity among myosins.

Local biosynthesis of corticosterone in rat skeletal muscle

Adrenal corticosterone plays crucial roles in energy metabolism and immuno-reactivity throughout the body. As we have previously shown that corticosterone biosynthesis in C2C12 myoblasts, we study about corticosterone biosynthesis in rat skeletal muscles. It was found that enzymatic activities producing corticosterone and testosterone except the activity of P450scc in rat skeletal muscle as like as C2C12 cells. The CYP11B mRNA encoding cytochrome P45011β that mediates 11-deoxycorticosterone hydroxylase activity, producing corticosterone was expressed in skeletal muscles. In immunoblotting analysis, cytochrome P45011β protein was expressed in rat muscles and whole organs especially higher levels in adrenal and brain. The localizations of corticosterone content and enzymatic activities involved in the production of corticosterone were preferentially observed in gastrocnemius fibers rather than in soleus fibers. The immunohistochemical analysis showed that the fast-twitch or type II muscle fibers positive to antibody against fast myosin heavy chain were preferentially stained with anti-cytochrome P45011β antibody in the gastrocnemius fiber. In addition, we detected corticosterone biosynthesis from pregnenolone sulfate conjugates in perfusion of the rat hindquarter. Corticosterone is synthesized in rat skeletal muscles and the biosynthesis was localized in the fast-twitch or type II muscle fibers. We speculated that the local synthesized corticosterone might be involved in glucocorticoid-induced muscle atrophy that preferentially occurs in fast muscle fibers, and the initial substrate of the local CORT biosynthesis were supported to be performed from the conjugates such as pregnenolone sulfate circulating in the blood flow.

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.

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.

Timing and Load-dependence of the Powerstroke and Pi-release in Skeletal Muscle Myosin

Muscle myosin converts chemical energy from ATP into mechanical work to power contraction, however it is still unclear how this transduction occurs at the molecular level. A key unanswered question is whether phosphate release precedes the powerstroke or if the powerstroke occurs first. This has been a challenging question to answer because these events occur faster than the time resolution of most instruments. Therefore, we used an ultrafast single molecule laser trap assay, with sub-millisecond time resolution, to directly visualize the timing of the powerstorke in fast skeletal muscle myosin. By elevating the level of phosphate in the buffer (15 mM) under varying ATP concentration, we ensured that myosin remained in a phosphate (Pi) bound state. We observed that myosin generated a powerstroke in the presence of phosphate that was similar in size (∼5 nm) and rate (∼5000 s−1 at 3 pN resistive force) to that observed in the absence of phosphate. This suggests that myosin generates its powerstroke prior to the release of phosphate. Further analyses are under way to determine if elevated levels of phosphate induced reversal of the powerstroke later in the binding event. Additional experiments were done over a range of assistive and resistive loads, from −6pN to +6pN, to determine the load-dependence of the powerstroke and the effect on myosin detachment rate from different chemomechanical states. Thus these data provide novel insights into the fundamental nature of force generation by muscle myosin.

Measuring quantitative proteomic distance between Spanish beef breeds

Estimates of quantitative proteomic distance between populations have not been reported to date. Here, quantitative proteomic distances between three Spanish bovine breeds (Asturiana de los Valles, AV; Retinta, RE; and Rubia Gallega, RG) were estimated from two-dimensional electrophoresis profiles of meat samples of longissimus thoracis muscle at 2 h post-mortem. Statistically significant distances were detected between AV/RG and the most genetically different RE breed, using the novel QD measure of quantitative proteomic distance. In total, 18 differentially abundant myofibrillar and sarcoplasmic proteins/isoforms contributing to proteomic distances between breeds were confidently identified by tandem mass spectrometry. The fast skeletal myosin regulatory light chain 2 followed by other five interacting proteins exhibited the most pronounced relative change between breeds. In addition, most differentially represented proteins could be associated with variations in meat tenderness. Therefore, they could be candidate biomarkers for molecular breeding programs and authentication of the three Spanish beef breeds.

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.

Assessment of muscle fiber adaptation in footballers using a new ELISA assay of myosin isoforms.

To measure the impact of training models on injury incidence, data of health and performance were integrated to study fiber adaptation during a competitive season. We studied football players over a season, analyzing hours of exposure to sport by serum changes in fast and slow myosin, creatine kinase and lactate dehydrogenase. A new assay was developed to measure the myosin isoforms in 49 non-sporting volunteers and in 27 professional football players. Myosin isoforms in volunteers with mean ages of 30±8 were 1553 µg/L fast and 1284 µg/L slow; in the group with of 56±7 were 1426 µg/L fast and 1046 µg/L slow. Slow myosin was significantly lower in older subjects (-18%). Samples from the players in preseason had lower mean scores for fast myosin (1123 µg/L) and higher for slow myosin (2072 µg/L) than reference volunteers. During the season, myosins reached the maximum with the maximum load (1537 µg/L fast, 2195 µg/L slow but decreased and adapted to the high level of demand (425 µg/L fast, 1342 µg/L slow). CK and LDH were maximal at the pre-season (227 U/L, 333 U/L) while myosin levels were maximal at the beginning of season (1537 µg/L, 2195 µg/L). Measuring serum myosin isoforms we identify the type and amount of damage caused by training and matches, making it a new control tool capable of advising training towards a minimum of blood slow myosin but controlling the fast fiber participating and be able to improve the performance of the players.

ITRAQ-based proteomic analysis reveals key proteins affecting cardiac function in broilers that died of sudden death syndrome

Sudden death syndrome (SDS), which is a cardiac-related condition commonly observed in chickens selected for rapid growth, causes significant economic losses to the global poultry industry. Its pathogenesis in broilers is poorly understood, and little is known about the proteome of the heart tissue of SDS broilers. A quantitative proteomic approach using isobaric tags for relative and absolute quantification labeling of peptides was used to characterize the protein expression profiles in the left ventricle of SDS broilers. These proteins were further analyzed by bioinformatics, and two proteins were validated by western blot analysis. We identified 186 differentially expressed proteins (DEPs), of which 72 were upregulated, and 114 were downregulated in the SDS group. Functional annotation suggested that 7 DEPs were related to cardiac muscle contraction, and another 7 DEPs were related to cardiac energy metabolism. Protein interaction network predictions indicated that differences in cardiac muscle contraction between SDS and healthy groups were regulated by troponin T, tropomyosin alpha-1 chain, fast myosin heavy chain HCIII, myosin-1B, coronin, and myoglobin, whereas differences in cardiac energy metabolism and biosynthesis of amino acids were regulated by gamma-enolase, phosphoglycerate mutase, NADH-ubiquinone oxidoreductase chain 2, serine/threonine-protein kinase, myoglobin, and alpha-amylase. Our expression profiles provide useful information and new insights into key proteins to elucidate SDS for further studies.

PQQ ameliorates skeletal muscle atrophy, mitophagy and fiber type transition induced by denervation via inhibition of the inflammatory signaling pathways.

Skeletal muscle atrophy involves and requires widespread changes in skeletal muscle gene expression and signaling pathway, resulting in excessive loss of muscle mass and strength, which is associated with poor prognosis and the decline of life quality in several diseases. However, the treatment of skeletal muscle atrophy remains an unresolved challenge to this day. The aim of the present study was to investigate the influence of pyrroloquinoline quinone (PQQ), a redox-active o-quinone found in various foods and mammalian tissues, on skeletal muscle atrophy, and to explore the underlying molecular mechanism. After denervation, mice were injected intraperitoneally with saline plus PQQ (5 mg/kg/d) or saline only for 14 days. The level of inflammatory cytokines in tibialis anterior (TA) muscles was determined by quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA), and the level of signaling proteins of Janus kinase 2/signal transduction and activator of transcription 3 (Jak2/STAT3), TGF-β1/Smad3, JNK/p38 MAPK, and nuclear factor κB (NF-κB) signaling pathway were detected by Western blot. The skeletal muscle atrophy was evaluated by muscle wet weight ratio and cross-sectional areas (CSAs) of myofibers. The mitophagy was observed through transmission electron microscopy (TEM) analysis, and muscle fiber type transition was analyzed through fast myosin skeletal heavy chain antibody staining. The proinflammatory cytokines IL-6, IL-1β and TNF-α were largely induced in TA muscles after sciatic nerve transection. PQQ can significantly reverse this phenomenon, as evidenced by the decreased levels of proinflammatory cytokines IL-6, IL-1β and TNF-α. Moreover, PQQ could significantly attenuate the signal activation of Jak2/STAT3, TGF-β1/Smad3, JNK/p38 MAPK, and NF-κB in skeletal muscles after sciatic nerve transection. Furthermore, PQQ alleviated skeletal muscle atrophy, mitigated mitophagy and inhibited slow-to-fast muscle fiber type transition. These results suggested that PQQ could attenuate denervation-induced skeletal muscle atrophy, mitophagy and fiber type transition through suppressing the Jak2/STAT3, TGF-β1/Smad3, JNK/p38 MAPK, and NF-κB signaling pathways.

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.

Sema3a-Nrp1 Signaling Mediates Fast-Twitch Myofiber Specificity of Tw2+ Cells.

We previously identified a unique population of interstitial muscle progenitors, marked by expression of the Twist2 transcription factor, which fuses specifically to type IIb/x fast-twitch myofibers. Tw2+ progenitors are distinct from satellite cells, a muscle progenitor that expresses Pax7 and contributes to all myofiber types. Through RNA sequencing and immunofluorescence, we identify the membrane receptor, Nrp1, as a marker of Tw2+ cells but not Pax7+ cells. We also found that Sema3a, a chemorepellent ligand for Nrp1, is expressed by type I and IIa myofibers but not IIb myofibers. Using stripe migration assays, chimeric cell-cell fusion assays, and a Sema3a transgenic mouse model, we identify Sema3a-Nrp1 signaling as a major mechanism for Tw2+ cell fiber-type specificity. Our findings reveal an extracellular signaling mechanism whereby a cell-surface receptor for a chemorepellent confers specificity of intercellular fusion of a specific muscle progenitor with its target tissue.

Muscle-specific gene expression and metabolic enzyme activities in Atlantic salmon Salmo salar L. fry reared under different photoperiod regimes.

This study was conducted to characterise the muscle-specific gene expression, energy metabolism level and growth rates of Atlantic salmon Salmo salar L. reared under different photoperiod regimes. The effects of two photoperiod regimes - LD 16:8 (16 h light:8 h dark) and LD 24:0 (24 h light:0 h dark) over a period of 3 months (August to October) on growth, energy metabolism enzyme activities (cytochrome c oxidase, COX; lactate dehydrogenase, LDH; and aldolase) and the gene expression levels of myogenic regulatory factors (MRFs - MyoD1 paralogues (MyoD1a, MyoD1b, MyoD1c), Myf5, MyoG), myostatin paralogues (MSTN-1a, MSTN-1b, MSTN-2a) and the fast skeletal myosin heavy chain (MyHC) in the muscles of Atlantic salmon underyearling fry (0+) were investigated. The experiment was conducted in a fish hatchery with natural variations in water temperature. The results were compared with those obtained in salmon reared under the lighting conditions of a fish hatchery (HL, hatchery lighting). The results revealed that the fry reared under constant light (LD 24:0) grew faster and were bigger at the end of the experiment. Fishes reared within the photoperiod regime LD 16:8 had a lower growth rate. COX activity was lower in fish under the LD 16:8 regime compared with the LD 24:0 group. The LDH and aldolase enzyme activities were higher in the group with constant light in comparison to control in the beginning of September. The expression level for all of the genes studied variated during the duration of the experiment, and MyHC, MyoG, MyoD1a and Myf5 expression depended on the light regime as well. The more noticeable changes in gene expression occurred in October. The MyHC and MyoG mRNA levels increased, accompanied by MyD1c gene expression, in both groups that had additional lighting (LD 16:8 and LD24:0) at the beginning of October and were higher than the HL group. In the HL group, the elevation of MyHC and MyoG mRNA was gradual during October, but there was a sharp increase in Myf5 expression at the beginning of October. MyoD1 paralogues differently expressed during the experiment. The MyoD1a mRNA level was elevated at the end of October along with MyHC and MyoG expression, but MyoD1b and MyoD1c mRNA levels decreased along with Myf5 gene expression. The expression of MSTN paralogues were elevated with increases in MyHC and MRFs transcripts. These findings show that constant light has a positive effect on the growth rate of salmon, affecting the aerobic and anaerobic capacity in their muscles. The alterations in muscle-specific gene expression between the groups with different light indicated that the mechanisms for regulating muscle growth processes in fish depend on photoperiod duration.

The first evidence of global meat phosphoproteome changes in response to pre-slaughter stress

BackgroundPre-slaughter stress (PSS) impairs animal welfare and meat quality. Dark, firm and dry (DFD) are terms used to designate poor quality meats induced by PSS. Protein phosphorylation can be a potentially significant mechanism to explain rapid and multiple physiological and biochemical changes linked to PSS-dependent muscle-to-meat conversion. However, the role of reversible phosphorylation in the response to PSS is still little known. In this study, we report a comparative phosphoproteomic analysis of DFD and normal meats at 24 h post-mortem from the longissimus thoracis (LT) bovine muscle of male calves of the Rubia Gallega breed. For this purpose, two-dimensional gel electrophoresis (2-DE), in-gel multiplex identification of phosphoproteins with PRO-Q Diamond phosphoprotein-specific stain, tandem (MALDI-TOF/TOF) mass spectrometry (MS), novel quantitative phosphoproteomic statistics and bioinformatic tools were used.ResultsNoticeable and statistically significant differences in the extent of protein phosphorylation were detected between sample groups at the qualitative and quantitative levels. Overall phosphorylation rates across significantly changed phosphoproteins were about three times higher in DFD than in normal meat. Significantly changed phosphoproteins involved a variable number of isoforms of 13 myofibrillar and sarcoplasmic nonredundant proteins. However, fast skeletal myosin light chain 2 followed by troponin T, F-actin-capping and small heat shock proteins showed the greatest phosphorylation change, and therefore they were the most important phosphoproteins underlying LT muscle conversion to DFD meat in the Rubia Gallega breed.ConclusionsThis is the first study reporting global meat phosphoproteome changes in response to PSS. The results show that reversible phosphorylation is a relevant mechanism underlying PSS response and downstream effects on meat quality. This research opens up novel horizons to unravel the complex molecular puzzle underlying muscle-to-meat conversion in response to PSS.

Poultry by-product meal as an alternative to fish meal in the juvenile gilthead seabream (Sparus aurata) diet

The aim of the present study was to evaluate the eventual possibility of fish meal (FM) substitution in the diet of farmed gilthead seabream (Sparus aurata) by poultry by-product meal (PBM) and to verify PBM suitability in aquafeeds. Inclusion of PBM in the seabream diet was therefore investigated by a multidisciplinary analysis to evaluate its possible effects on fish growth performance, fish welfare and fillet quality. A control diet and two experimental diets, Feed A and Feed B containing PBM with 50% and 100% of FM substitution respectively, were formulated. All diets were isoproteic (45%), isolipidic (20%) and isoenergetic. The growth trial lasted 110 days, including 20 days of fish acclimatization. Juvenile gilthead seabream with an initial average weight of 73.57 ± 10.47 g were allotted at random in nine tanks (three replicates per diet), fed once a day by hand (feeding rate 1%).As a result, average weight gain increased in all fish groups without any statistically significant difference (P > .05). Measured zootechnical parameters were similar among fish groups, the condition factor as an indicator of fish condition was around 2 and the survival rate was 100%. Investigations through haematological parameters, digestive enzymes and liver histology analyses demonstrated that no statistical difference existed among dietary treatments and this clear evidence attests that PBM inclusion in seabream diets did not have a negative effect on fish welfare. Protein patterns obtained from fish fed the control diet and PBM diets showed a similar expression of structural proteins such as actin, tropomyosin, myosin light chain 1 (MLC1), myosin light chain 2 (MLC2) and fast skeletal myosin light chain (MLC3). Results for fish fillet compositions were comparable in all fish groups with some exceptions for fatty acid composition. The gross energy content of seabream muscle was also not affected by PBM.The present study demonstrated that the total substitution of FM with PBM in the commercial diet of gilthead seabream (S. aurata) is achievable without compromising fish growth performance, fish welfare and fillet quality and suggests that PBM could be considered as a good sustainable raw material for fish food.

Testosterone Supplementation Enhances Lean Body Mass But Not Lower-Body Muscular Strength and Endurance During Severe Exercise- and Diet-Induced Energy Deficit

Sustained periods of severe energy deficit during military operations, produced by significant increases in aerobic-type physical work and limited dietary intake, result in conditions that degrade lean body mass (LBM) and lower-body muscle function, which may be mediated by concomitant reductions in circulating testosterone. Therefore, we studied the effects of restoring testosterone by supplementation (200 mg testosterone enanthate/wk, TEST), compared to placebo (1 mL sesame seed oil/wk, PLA), on body composition and lower-body muscular strength and endurance during 28 d of severe exercise- and diet-induced energy deficit in non-obese, young men. Following energy deficit, concentrations of free testosterone decreased, total testosterone remained unchanged, and sex-hormone binding globulin (SHBG) increased in PLA; whereas, total and free testosterone increased and SHBG was unchanged in TEST, such that, total and free testosterone were greater and SHBG concentrations were lower in TEST than PLA. During energy deficit, TEST attenuated weight loss and increased DXA-derived LBM compared to PLA, but both groups had a similar increase in type I slow twitch myosin heavy chain cross-sectional area (CSA) and decrease in type II fast twitch myosin heavy chain CSA. Lower-body muscle function declines and adverse event rates did not differ by group during energy deficit. Supplemental testosterone may be an effective pharmacological LBM enhancement strategy during short-term periods of exercise- and diet-induced energy deficit in non-obese, young men, but does not appear to mitigate lower-body functional decline. Funding:The research reported in this publication is supported by the Collaborative Research to Optimize Warfighter Nutrition II and III projects and the Joint Program Committee-5, funded by the US Department of Defense. Declaration of Interest: J.C.W., O.T.C., and K.M.G. reported that their institution received funding from the US Department of Defense for work associated with this publication. H.R.L. reported receiving personal fees from Pfizer, Inc., for work outside this publication. All remaining authors declare no competing interests. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Army or the Department of Defense. Any citations of commercial organizations and trade names in this report do not constitute an official Department of the Army endorsement of approval of the products or services of these organizations. Ethical Approval: This study was approved by the Pennington Biomedical Research Center (PBRC) Institutional Review Board and the Human Research Protection Office of the US Army Medical Research and Materiel Command.