Fast Myosin Heavy Chain Research Articles

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.

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.

Resveratrol regulates muscle fiber type conversion via miR-22-3p and AMPK/SIRT1/PGC-1α pathway

This study investigated the effects of resveratrol and miR-22-3p on muscle fiber type conversion in mouse C2C12 myotubes. Here we showed that resveratrol significantly increased the protein level of slow myosin heavy chain (MyHC) and the activities of succinic dehydrogenase and malate dehydrogenase, as well as markedly decreased the protein level of fast MyHC and the activity of lactate dehydrogenase. Immunofluorescence staining showed that resveratrol remarkably upregulated the number of slow MyHC-positive myotubes and downregulated the number of fast MyHC-positive myotubes, suggesting that resveratrol promoted muscle fiber type conversion from fast-twitch to slow-twitch in C2C12 myotubes. We also showed that miR-22-3p had an opposite function on muscle fiber type conversion and resveratrol was able to repress the expression of miR-22-3p. Furthermore, AMP-activated protein kinase (AMPK) inhibitor Compound C and miR-22-3p mimics could attenuate and eliminate muscle fiber type conversion from fast-twitch to slow-twitch cause by resveratrol, respectively. Together, we provided the first evidence that resveratrol promotes muscle fiber type conversion from fast-twitch to slow-twitch via miR-22-3p and AMPK/SIRT1/PGC-1α pathway in C2C12 myotubes.

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.

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.

Characterisation of flexor digitorum profundus, flexor digitorum superficialis and extensor digitorum communis by electrophoresis and immunohistochemical analysis of myosin heavy chain isoforms in older men

IntroductionFew data exist on the fiber type composition of the extrinsic finger muscles. The aim of the present study was to describe myosin heavy chain (MyHC) composition of flexor digitorum profundus (FDP), flexor digitorum superficialis (FDS) and extensor digitorum communis (EDC). MyHC composition is relevant for whole muscle contractile performance and several studies on single muscle fibers demonstrated that fibers expressing only slow MyHC-1 develop less specific force than fibers expressing fast MyHCs. Since contraction force of finger extensors is smaller than of finger flexors a hypothesis was posited that the content of MyHC-1 is higher in EDC than in extrinsic finger flexors. MethodsAutopsy samples of FDP, FDS, and EDC in 27 healthy older men were analyzed and compared with each other and with biceps brachii (BB). MyHC isoforms were quantified on silver-stained 6% SDS-PAGE. Muscle fibers were classified immunohistochemically according to the expression of adult MyHC isoforms and their morphometric parameters were determined. ResultsEDC stood out for its higher proportion of slow MyHC-1 (50%) compared to FDP (37%), FDS (38%) and BB (35%) (p<0.001 in all), and its lower proportion of fast MyHC-2x (13%) compared to FDP (26%, p=0.001), FDS (22%, p=0.028) and BB (29%, p<0.001) detected on SDS-PAGE. Immunohistochemically, EDC had a higher area proportion of pure slow type-1 fibers (63%) than FDP (47%, p=0.002), FDS (49%, p=0.007) and BB (47%, p=0.002), and lower area proportion of pure fast type-2x fibers (2%) than FDP (12%, p=0.014), FDS (8%, p=0.256) and BB (14%, p=0.002). All muscles contained a similar area proportion of pure type-2a fibers and hybrid type-2a/2x fibers. ConclusionsThe study results support the hypothesis that the content of MyHC-1 is higher in EDC than in extrinsic finger flexors, which is in agreement with the lower contraction force of finger extensors compared to finger flexors.

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.

Histone deacetylase activity mediates thermal plasticity in zebrafish (Danio rerio)

Regulatory mechanisms underlying thermal plasticity determine its evolution and potential to confer resilience to climate change. Here we show that class I and II histone deacetylases (HDAC) mediated thermal plasticity globally by shifting metabolomic profiles of cold acclimated zebrafish (Danio rerio) away from warm acclimated animals. HDAC activity promoted swimming performance, but reduced slow and fast myosin heavy chain content in cardiac and skeletal muscle. HDAC increased sarco-endoplasmic reticulum ATPase activity in cold-acclimated fish but not in warm-acclimated animals, and it promoted cardiac function (heart rate and relative stroke volume) in cold but not in warm-acclimated animals. HDAC are an evolutionarily ancient group of proteins, and our data show that they mediate the capacity for thermal plasticity, although the actual manifestation of plasticity is likely to be determined by interactions with other regulators such as AMP-activated protein kinase and thyroid hormone.

Muscle growth mechanisms in response to isoenergetic changes in dietary non-protein energy source at low and high protein levels in juvenile rainbow trout

This study investigates muscle growth mechanisms in juvenile rainbow trout in response to isoenergetic changes in dietary non-protein energy (NPE) source (F, fat vs. C, carbohydrates) at two levels of digestible protein to digestible energy (DP/DE) ratio. Fish (initial weight 32.4 g) were fed four diets having similar DE levels (~18 kJ g−1) with a high (HP/E~26 mg kJ−1) vs. low (LP/E~14 mg kJ−1) DP/DE ratio using F or C as major NPE source (7 week-experiment). The lowering of dietary DP/DE ratio increased myoblast determination protein 1a (myod1a) and decreased myostatin 1b (mstn1b) and cathepsin D (ctsd) muscle mRNA levels. The isoenergetic change in dietary NPE from F to C decreased myod1a and proliferative cell nuclear antigen (pcna) muscle mRNA levels. An interaction between DP/DE ratio and NPE source was observed in muscle transcript levels of myogenic factor 6 (mrf4/myf6), fast myosin heavy chain (fmhc) and fast myosin light chain 2 (fmlc2). White muscle total cross-sectional area decreased at low dietary DP/DE ratio and also when NPE source changed from F to C, linked i) to a decreased total number of white muscle fibres, indicating that low dietary DP/DE restricted muscle hyperplasia and that dietary carbohydrate were less efficiently used than fat to sustain muscle hyperplasia, and ii) to decreased percentage of large muscle fibres, indicating limited fibre hypertrophy. Not only the DP level or the DP/DE ratio, but also the isoenergetic change in dietary NPE source (fat vs carbohydrates) thus appears as a potent regulator of muscle hyperplasia and hypertrophy.

Duplication of a Single myhz1.1 Gene Facilitated the Ability of Goldfish (Carassius auratus) to Alter Fast Muscle Contractile Properties With Seasonal Temperature Change.

Seasonal temperature changes markedly effect the swimming performance of some cyprinid fish acutely tested at different temperatures, involving a restructuring of skeletal muscle phenotype including changes in contractile properties and myosin heavy chain expression. We analyzed the transcriptome of fast myotomal muscle from goldfish (Carassius auratus L.) acclimated to either 8 or 25°C for 4 weeks (12 h light: 12 h dark) and identified 10 myosin heavy chains (myh) and 13 myosin light chain (myl) transcripts. Goldfish orthologs were classified based on zebrafish nomenclature as myhz1.1α, myhz1.1β, myhz1.1γ, myha, myhb, embryo_myh1, myh9b, smyh2, symh3, and myh11 (myosin heavy chains) and myl1a, myl1b, myl2, myl9a, myl9b, myl3, myl13, myl6, myl12.1a, myl12.1b, myl12.2a, myl12.2b, and myl10 (myosin light chains). The most abundantly expressed transcripts myhz1.1α, myhz1.1β, myhz1.1γ, myha, myl1a, myl1b, myl2, and myl3) were further investigated in fast skeletal muscle of goldfish acclimated to either 4, 8, 15, or 30°C for 12 weeks (12 h light:12 h dark). Total copy number for the myosin heavy chains showed a distinct optimum at 15°C (P < 0.01). Together myhz1.1α and myhz1.1β comprised 90 to 97% of myhc transcripts below 15°C, but only 62% at 30°C. Whereas myhz1.1α and myhz1.1β were equally abundant at 4 and 8°C, myhz1.1β transcripts were 17 and 12 times higher than myhz1.1α at 15 and 30°C, respectively, (P < 0.01). Myhz1.1γ expression was at least nine-fold higher at 30°C than at cooler temperatures (P < 0.01). In contrast, the expression of myha and myosin light chains showed no consistent pattern with acclimation temperature. A phylogenetic analysis indicated that the previously reported ability of goldfish and common carp to alter contractile properties and myofibrillar ATPase activity with temperature acclimation was related to the duplication of a single myhz1.1 fast muscle myosin heavy chain found in basal cyprinids such as the zebrafish (Danio rerio).

Leucine promotes porcine myofibre type transformation from fast-twitch to slow-twitch through the protein kinase B (Akt)/forkhead box 1 signalling pathway and microRNA-27a.

Muscle fibre types can transform from slow-twitch (slow myosin heavy chain (MyHC)) to fast-twitch (fast MyHC) or vice versa. Leucine plays a vital effect in the development of skeletal muscle. However, the role of leucine in porcine myofibre type transformation and its mechanism are still unclear. In this study, effects of leucine and microRNA-27a (miR-27a) on the transformation of porcine myofibre type were investigated in vitro. We found that leucine increased slow MyHC protein level and decreased fast MyHC protein level, increased the levels of phospho-protein kinase B (Akt)/Akt and phospho-forkhead box 1 (FoxO1)/FoxO1 and decreased the FoxO1 protein level. However, blocking the Akt/FoxO1 signalling pathway by wortmannin attenuated the role of leucine in porcine myofibre type transformation. Over-expression of miR-27a decreased slow MyHC protein level and increased fast MyHC protein level, whereas inhibition of miR-27a had an opposite effect. We also found that expression of miR-27a was down-regulated following leucine treatment. Moreover, over-expression of miR-27a repressed transformation from fast MyHC to slow MyHC caused by leucine, suggesting that miR-27a is interdicted by leucine and then contributes to porcine muscle fibre type transformation. Our finding provided the first evidence that leucine promotes porcine myofibre type transformation from fast MyHC to slow MyHC via the Akt/FoxO1 signalling pathway and miR-27a.

Muscle fibre type composition in the lateral muscle of olive flounder Paralichthys olivaceus

In this paper, a combined-method study has been made on the lateral muscle of the teleost olive flounder Paralichthys olivaceus in just-hatched and adult stages. In just-hatched stage, both slow and fast muscle fibres were detected: (1) in situ hybridization analysis indicated that slow and fast myosin heavy chain genes were specifically expressed in the superficial and deep part of the myotomal muscle, respectively; (2) immunohistochemistry analysis showed that fibres in the deep part reacted with anti-fast myosin antibody F310; (3) western blot analysis detected a weak expression of slow myosin and a strong expression of fast myosin. In adult stage, the slow and fast muscle fibres had their own distribution characteristics: (1) hematoxylin/eosin staining showed the histological characteristics of the muscle fibre composition; (2) histochemical observations showed that the deep muscle fibres, and some fibres near the epidermis, contain alkali-stable myofibrillar ATPase activity; (3) immunohistochemistry analysis indicated that all the deep muscle fibres reacted with F310 antibody and some fibres in the superficial layer of muscle also reacted with F310; (4) western blot analysis showed that fast myosin was expressed both in the blended muscles (the mix of superficial and deep muscles) and deep muscles, while slow myosin was mainly expressed in the blended muscles. These findings suggested that both slow and fast muscle fibres existed in the musculature of the olive flounder in just-hatched and adult stages. Notably, the adult fast fibres also exist in the superficial layer of the muscle.

Arginine promotes skeletal muscle fiber type transformation from fast-twitch to slow-twitch via Sirt1/AMPK pathway

This study investigated the effect of arginine on skeletal muscle fiber type transformation in mice and in C2C12 myotubes. Our data showed that dietary supplementation of arginine in mice significantly up-regulated the slow myosin heavy chain (MyHC), troponin I-SS, sirtuin1 (Sirt1) and peroxisome proliferator activated receptor-γ coactivator-1α (PGC-1α) protein expressions, as well as significantly down-regulated the fast MyHC protein expression. In C2C12 myotubes, arginine significantly increased the protein level of slow MyHC and the number of slow MyHC-positive cells, as well as significantly decreased the protein level of fast MyHC and the number of fast MyHC-positive cells. We also showed that arginine increased the activities of succinic dehydrogenase and malate dehydrogenase and decreased the activity of lactate dehydrogenase in mice and in C2C12 myotubes. Here we found that AMP-activated protein kinase (AMPK) was activated by arginine in mice and in C2C12 myotubes. However, inhibition of AMPK activity by compound C significantly attenuated the effects of arginine on slow MyHC and fast MyHC expressions in C2C12 myotubes. Finally, we showed that inhibition of Sirt1 expression by EX527 attenuated arginine-induced increase in the protein levels of phospho-AMPK and slow MyHC, the mRNA level of nitric oxide synthase (NOS) and the contents of NOS and NO, as well as decrease in fast MyHC protein level. Together, our findings indicated that arginine promotes skeletal muscle fiber type switching from fast-twitch to slow-twitch via Sirt1/AMPK pathway.

Effect of sodium 4-phenylbutyrate on Clenbuterol-mediated muscle growth.

Previously, we highlighted induction of an integrated stress response (ISR) gene program in skeletal muscle of pigs treated with a beta-adrenergic agonist. Hence we tested the hypothesis that the ER-stress inhibitor, sodium 4-phenylbutyrate (PBA), would inhibit Clenbuterol-mediated muscle growth and reduce expression of genes that are known indicators of an ISR in mice. Clenbuterol (1mg/kg/day) administered to C57BL6/J mice for 21 days increased body weight (p<0.001), muscle weights (p<0.01), and muscle fibre diameters (p<0.05). Co-administration of PBA (100mg/kg/day) did not alter the Clenbuterol-mediated phenotype, nor did PBA alone have any effects compared to that of the vehicle treated mice. Clenbuterol increased skeletal muscle mRNA expression of phosphoserine amino transferase 1 (PSAT1, p<0.001) and cyclophillin A (p<0.01) at day 3, but not day 7. Clenbuterol decreased mRNA expression of activating transcription factor (ATF) 4 and ATF5 at day 3 (p<0.05) and day 7 (p<0.01), X-box binding protein 1 (XBP1) variant 2 mRNA at day 3 only (p<0.01) and DNA damage inducible transcript 3 (DDIT3/CHOP) mRNA at day 7 only (p<0.05). Co-administration of PBA had no effect on Clenbuterol-induced changes in skeletal muscle gene expression. In contrast, treatment of C2C12 myotubes with 5mM PBA (8hr) attenuated the thapsigargin-induced ISR gene program. Prolonged (24-48hr) treatment with PBA caused atrophy (p<0.01), reduced neoprotein synthesis (p<0.0001) and decreased expression of myogenin and fast myosin heavy chain genes (p<0.01), indicating an inhibition of myogenic differentiation. In summary, Clenbuterol did not induce an ISR gene program in mouse muscle. On the contrary, it reduced expression of a number of ISR genes, but it increased expression of PSAT1 mRNA. Co-administration of PBA had no effect on Clenbuterol-mediated muscle growth or gene expression in mice, whereas PBA did inhibit thapsigargin-induced ISR gene expression in cultured C2C12 cells and appeared to inhibit myogenic differentiation, independent of altering ISR gene expression.

Damaged muscle fibers might masquerade as hybrid fibers - a cautionary note on immunophenotyping mouse muscle with mouse monoclonal antibodies.

We report that, labeling mouse muscle tissue, with mouse monoclonal antibodies specific to slow or fast myosin heavy chain (sMyHC and fMyHC, respectively), can lead to artefactual labeling of damaged muscle fibers, as hybrid fibers (sMyHC+ and fMyHC+). We demonstrate that, such erroneous immunophenotyping of muscle may be avoided, by performing colabeling or serialsection- labeling, to identify damaged fibers. The quadriceps femorismuscle group (QF) in 7-month-old, male, C57BL/6J mice had: 1.21±0.21%, 98.34±1.06%, 0.07±0.01%, and 0.53±0.85% fibers, that were, sMyHC+, fMyHC+, hybrid, and damaged, respectively. All fibers in the tibialis anterior muscle (TA) of 3-month-old, male, C57BL/6J mice were fMyHC+; and at 3 days after injurious eccentric contractions, there was no fiber-type shift, but ~ 18% fibers were damaged.

Maximal strength training increases muscle force generating capacity and the anaerobic ATP synthesis flux without altering the cost of contraction in elderly.

Aging is associated with a progressive decline in skeletal muscle function, then leading to impaired exercise tolerance. Maximal strength training (MST) appears to be a practical and effective intervention to increase both exercise capacity and efficiency. However, the underlying physiological mechanisms responsible for these functional improvements are still unclear. Accordingly, the purpose of this study was to examine the intramuscular and metabolic adaptations induced by 8 weeks of knee-extension MST in the quadriceps of 10 older individuals (75 ± 9 yrs) by employing a combination of molecular, magnetic resonance 1H-imaging and 31P-spectroscopy, muscle biopsies, motor nerve stimulation, and indirect calorimetry techniques. Dynamic and isometric muscle strength were both significantly increased by MST. The greater torque-time integral during sustained isometric maximal contraction post-MST (P = 0.002) was associated with increased rates of ATP synthesis from anaerobic glycolysis (PRE: 10 ± 7 mM·min-1; POST: 14 ± 7 mM·min-1, P = 0.02) and creatine kinase reaction (PRE: 31 ± 10 mM·min-1; POST: 41 ± 10 mM·min-1, P = 0.006) such that the ATP cost of contraction was not significantly altered. Expression of fast myosin heavy chain, quadriceps muscle volume, and submaximal cycling net efficiency were also increased with MST (P = 0.005; P = 0.03 and P = 0.03, respectively). Overall, MST induced a shift toward a more glycolytic muscle phenotype allowing for greater muscle force production during sustained maximal contraction. Consequently, some of the MST-induced improvements in exercise tolerance might stem from a greater anaerobic capacity to generate ATP, while the improvement in exercise efficiency appears to be independent from an alteration in the ATP cost of contraction.

Effects of elevated H+ and Pi on the contractile mechanics of skeletal muscle fibres from young and old men: implications for muscle fatigue in humans.

The mechanisms responsible for the loss in muscle power and increased fatigability with ageing are unresolved. We show that the contractile mechanics of fibres from the vastus lateralis of old men were well-preserved compared to those of young men, but the selective loss of fast myosin heavy chain II muscle was strongly associated with age-related decrements in whole-muscle strength and power. We reveal that the combination of acidosis (H+ ) and inorganic phosphate (Pi ) is an important mediator of muscle fatigue in humans by inhibiting the low- to high-force state of the cross-bridge cycle and peak power, but the depressive effects of these ions on cross-bridge function were similar in fibres from young and old men. These findings suggest that the age-related loss in muscle power is primarily determined by the atrophy of fast fibres, but the age-related increased fatigability cannot be explained by an increased sensitivity of the cross-bridge to H+ and Pi . The present study aimed to identify the mechanisms responsible for the loss in muscle power and increased fatigability with ageing by integrating measures of whole-muscle function with single fibre contractile mechanics. After adjusting for the 22% smaller muscle mass in old (73-89years, n=6) compared to young men (20-29years, n=6), isometric torque and power output of the knee extensors were, respectively, 38% and 53% lower with age. Fatigability was ∼2.7-fold greater with age and strongly associated with reductions in the electrically-evoked contractile properties. To test whether cross-bridge mechanisms could explain age-related decrements in knee extensor function, we exposed myofibres (n=254) from the vastus lateralis to conditions mimicking quiescent muscle and fatiguing levels of acidosis (H+ ) (pH 6.2) and inorganic phosphate (Pi ) (30mm). The fatigue-mimicking condition caused marked reductions in force, shortening velocity and power and inhibited the low- to high-force state of the cross-bridge cycle, confirming findings from non-human studies that these ions act synergistically to impair cross-bridge function. Other than severe age-related atrophy of fast fibres (-55%), contractile function and the depressive effects of the fatigue-mimicking condition did not differ in fibres from young and old men. The selective loss of fast myosin heavy chain II muscle was strongly associated with the age-related decrease in isometric torque (r=0.785) and power (r=0.861). These data suggest that the age-related loss in muscle strength and power are primarily determined by the atrophy of fast fibres, but the age-related increased fatigability cannot be explained by an increased sensitivity of the cross-bridge to H+ and Pi .

P-246 - Distinct proteasomal activity for fast and slow twitch skeletal muscle during aging

Skeletal muscle alterations during aging lead to dysfunctional metabolism, correlating with frailty and early mortality. The loss of proteostasis is a hallmark of aging. Whether proteostasis loss plays a role in muscle aging remains elusive. To address this question we collected muscles, Soleus (SOL, type I) and Extensor digitorum longus (EDL, type II), from young (4 months) and old (25 months) C57BL/6 mice and evaluated the proteasomal system. Initial work showed decreased 26 S activity in old SOL. EDL displayed lower proteasomal activity in both ages compared to any of the SOL ages. Moreover, in order to understand if during aging there is the so-called “fiber switch from fast-to-slow”, we performed western blots against sMHC and fMHC (slow and fast myosin heavy chain, respectively). Preliminary results suggest that young SOL is composed by slow twitch fibers but also contains fast twitch fibers, while young EDL seems to be mostly composed by fast twitch fibers that level down during aging, suggesting the switch. As a conclusion, EDL seems to have less proteasomal activity, however, if this is a contributor or a consequence to the muscle fiber switch during aging still needs further investigation.

Soleus muscle stability in wild hibernating black bears.

Based on studies of fast skeletal muscles, hibernating black and brown bears resist skeletal muscle atrophy during months of reduced physical activity and not feeding. The present study examined atrophy sparing in the slow soleus muscle, known to be highly prone to disuse atrophy in humans and other mammals. We demonstrated histochemically that the black bear soleus is rich in slow fibers, averaging 84.0 ± 6.6%. The percentages of slow fibers in fall (87.3 ± 4.9%) and during hibernation (87.1 ± 5.6%) did not differ ( P = 0.3152) from summer. The average fiber cross-sectional area to body mass ratio (48.6 ± 11.7 µm2/kg) in winter hibernating bears was not significantly different from that of summer (54.1 ± 11.8 µm2/kg, P = 0.4186) and fall (47.0 ± 9.7 µm2/kg, P = 0.9410) animals. The percentage of single hybrid fibers containing both slow and fast myosin heavy chains, detected biochemically, increased from 2.6 ± 3.8% in summer to 24.4 ± 24.4% ( P = 0.0244) during hibernation. The shortening velocities of individual hybrid fibers remained unchanged from that of pure slow and fast fibers, indicating low content of the minority myosins. Slow and fast fibers in winter bears exhibited elevated specific tension (kN/m2; 22%, P = 0.0161 and 11%, P = 0.0404, respectively) and maintained normalized power. The relative stability of fiber type percentage and size, fiber size-to-body mass ratio, myosin heavy chain isoform content, shortening velocity, power output, and elevated specific tension during hibernation validates the ability of the black bear to preserve the biochemical and performance characteristics of the soleus muscle during prolonged hibernation.

Changes in Myosin Heavy Chain Isoforms Along the Length of Orbital Fibers in Rabbit Extraocular Muscle.

Extraocular muscles express 10 myosin heavy chain (MyHC) isoforms that cater for a wide range of contractile speeds. We aim to characterize the variations in MyHC expression along the length of singly (SIFs) and multiply innervated fibers (MIFs) in the orbital layer of rabbit superior rectus muscle. Monospecific antibodies to nine MyHCs, including an anti-slow-tonic antibody characterized here were used to immunohistochemically map variations in MyHC distribution in serial sections along the muscle's full length. The fastest MyHC, EO, is expressed at the endplate zone (EPZ) of SIFs, flanked proximally and distally by segments expressing the slower 2A, with or without embryonic MyHC. MIFs with constant diameter express α-cardiac MyHC at the EPZ, flanked by segments co-expressing α-cardiac/embryonic and possibly slow-tonic MyHCs. MIFs with varying diameter also express α-cardiac MyHC at the EPZ in their thin, central region, flanked by thin segments co-expressing α-cardiac/embryonic MyHCs, with long proximal and distal extensions of larger diameter that co-express embryonic/slow-tonic and α-cardiac or β/slow MyHCs. Orbital fiber types express multiple MyHCs, with faster ones in SIFs, slower ones in MIFs, but all have fast EPZs and slower end segments. We hypothesize that these unique MyHC distributions enable these fibers to relax in two kinetically distinct phases while acting in an antagonistic manner during a saccade: the fast phases facilitate acceleration of eyeball rotation during agonist contraction, while the slow phases help its deceleration toward the visual target, thereby linearizing the saccade. These properties also facilitate pulley movements to implement Listing's law.