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1.  Mechanosensing in Myosin Filament Solves a 60 Years Old Conflict in Skeletal Muscle Modeling between High Power Output and Slow Rise in Tension 
Almost 60 years ago Andrew Huxley with his seminal paper (Huxley, 1957) laid the foundation of modern muscle modeling, linking chemical to mechanical events. He described mechanics and energetics of muscle contraction through the cyclical attachment and detachment of myosin motors to the actin filament with ad-hoc assumptions on the dependence of the rate constants on the strain of the myosin motors. That relatively simple hypothesis is still present in recent models, even though with several modifications to adapt the model to the different experimental constraints which became subsequently available. However, already in that paper, one controversial aspect of the model became clear. Relatively high attachment and detachment rates of myosin to the actin filament were needed to simulate the high power output at intermediate velocity of shortening. However, these rates were incompatible with the relatively slow rise in tension upon activation, despite the rise should be generated by the same rate functions. This discrepancy has not been fully solved till today, despite several hypotheses have been forwarded to reconcile the two aspects. Here, using a conventional muscle model, we show that the recently revealed mechanosensing mechanism of recruitment of myosin motors (Linari et al., 2015) can solve this long standing problem without any further ad-hoc hypotheses.
doi:10.3389/fphys.2016.00427
PMCID: PMC5034546  PMID: 27721796
muscle; modeling; super-relaxed state; myosin motors; power output
2.  ANTIOXIDANTS PROTECT CALSEQUESTRIN-1 KNOCKOUT MICE FROM HALOTHANE- AND HEAT-INDUCED SUDDEN DEATH 
Anesthesiology  2015;123(3):603-617.
Background
Mice lacking calsequestrin-1 (CASQ1-null), a Ca2+ binding protein that modulates the activity of Ca2+ release in skeletal muscle, exhibit lethal hypermetabolic episodes that resemble malignant hyperthermia (MH) in humans when exposed to halothane or heat stress.
Methods
As oxidative species may play a critical role in MH crises, we treated CASQ1-null mice with two antioxidants, N-acetylcysteine (NAC provided ad libitum in drinking water) and Trolox (administered by intra-peritoneal injection), before exposure to halothane (2%, 1 h) or heat (41°C, 1 h).
Results
NAC and Trolox significantly protected CASQ1-null mice from lethal episodes, with mortality being: 79% (n=14), 25% (n=16) and 20% (n=5) during halothane exposure and 86% (n=21), 29% (n=21) and 33% (n=6) during heat-stress in un-treated, NAC- and Trolox- treated mice, respectively. During heat challenge, the increase in core temperature in CASQ1-null mice (42.3±0.1°C, n=10) was significantly reduced by both NAC and Trolox (40.6±0.3°C, n=6; 40.5±0.2°C, n=6). NAC treatment of CASQ1-null muscles/mice normalized caffeine sensitivity during in-vitro contracture tests, Ca2+ transients in single fibers, and significantly reduced the percentage of fibers undergoing rhabdomyolysis (37.6±2.5%, 38/101 fibers in 3 mice; 11.6±1.1%, 21/186 fibers in 5 mice respectively). The protective effect of antioxidant treatment likely resulted from mitigation of oxidative stress, as NAC reduced mitochondrial superoxide production, superoxide dismutase type-1 (SOD1) and 3-nitrotyrosine (3-NT) expression, and increased both reduced glutathione (GSH) and GSH/GSSG ratio.
Conclusions
These studies provide a deeper understanding of the mechanisms that underlie hyperthermic crises in calsequestrin-1 deficient muscle and demonstrate that antioxidant pre-treatment may prevent them.
doi:10.1097/ALN.0000000000000748
PMCID: PMC4543432  PMID: 26132720
3.  Spectroscopic Studies of the Super Relaxed State of Skeletal Muscle 
PLoS ONE  2016;11(8):e0160100.
In the super-relaxed state of myosin, ATPase activity is strongly inhibited by binding of the myosin heads to the core of the thick filament in a structure known as the interacting-heads motif. In the disordered relaxed state myosin heads are not bound to the core of the thick filament and have an ATPase rate that is 10 fold greater. In the interacting-heads motif the two regulatory light chains appear to bind to each other. We have made single cysteine mutants of the regulatory light chain, placed both paramagnetic and fluorescent probes on them, and exchanged them into skinned skeletal muscle fibers. Many of the labeled light chains tended to disrupt the stability of the super-relaxed state, and showed spectral changes in the transition from the disordered relaxed state to the super-relaxed state. These data support the putative interface between the two regulatory light chains identified by cryo electron microscopy and show that both the divalent cation bound to the regulatory light chain and the N-terminus of the regulatory light chain play a role in the stability of the super-relaxed state. One probe showed a shift to shorter wavelengths in the super-relaxed state such that a ratio of intensities at 440nm to that at 520nm provided a measure of the population of the super-relaxed state amenable for high throughput screens for finding potential pharmaceuticals. The results provide a proof of concept that small molecules that bind to this region can destabilize the super-relaxed state and provide a method to search for small molecules that do so leading to a potentially effective treatment for Type 2 diabetes and obesity.
doi:10.1371/journal.pone.0160100
PMCID: PMC4968846  PMID: 27479128
4.  The Regenerative Potential of Female Skeletal Muscle upon Hypobaric Hypoxic Exposure 
Aim: The aim of this study was to determine whether a 14-day trekking expeditions, in high altitude hypoxic environment, triggers redox disturbance at the level of satellite cells (adult stem cells) in young women.
Methods: We collected muscle biopsies from Vastus Lateralis muscle for both single fiber analysis and satellite cells isolation. The samples collected before (PRE-Hypoxia) and after (POST-Hypoxia) the trekking in the Himalayas were compared. Satellite cells were investigated for oxidative stress (oxidant production, antioxidant enzyme activity, and lipid damage), mitochondrial potential variation, gene profile of HIF, and myogenic transcription factors (Pax7, MyoD, myogenin), and miRNA expression (miR-1, miR-133, miR-206).
Results: The nuclear domain analysis showed a significant fusion and consequent reduction of the Pax7+ satellite cells in the single mature fibers. The POST-Hypoxia myoblasts obtained by two out of six volunteers showed high superoxide anion production and lipid peroxidation along with impaired dismutase and catalase and mitochondrial potential. The transcription profile and miRNA expression were different for oxidized and non-oxidized cells.
Conclusions: The present study supports the phenomenon of hypobaric-hypoxia-induced oxidative stress and its role in the impairment of the regenerative capacity of satellite cells derived from the V. Lateralis muscle of young adult female subjects.
doi:10.3389/fphys.2016.00303
PMCID: PMC4943944  PMID: 27471475
hypoxia; miRNA; oxidative stress; satellite cells; single fiber; women
5.  Protein Supplementation Does Not Further Increase Latissimus Dorsi Muscle Fiber Hypertrophy after Eight Weeks of Resistance Training in Novice Subjects, but Partially Counteracts the Fast-to-Slow Muscle Fiber Transition 
Nutrients  2016;8(6):331.
The response to resistance training and protein supplementation in the latissimus dorsi muscle (LDM) has never been investigated. We investigated the effects of resistance training (RT) and protein supplementation on muscle mass, strength, and fiber characteristics of the LDM. Eighteen healthy young subjects were randomly assigned to a progressive eight-week RT program with a normal protein diet (NP) or high protein diet (HP) (NP 0.85 vs. HP 1.8 g of protein·kg−1·day−1). One repetition maximum tests, magnetic resonance imaging for cross-sectional muscle area (CSA), body composition, and single muscle fibers mechanical and phenotype characteristics were measured. RT induced a significant gain in strength (+17%, p < 0.0001), whole muscle CSA (p = 0.024), and single muscle fibers CSA (p < 0.05) of LDM in all subjects. Fiber isometric force increased in proportion to CSA (+22%, p < 0.005) and thus no change in specific tension occurred. A significant transition from 2X to 2A myosin expression was induced by training. The protein supplementation showed no significant effects on all measured outcomes except for a smaller reduction of 2X myosin expression. Our results suggest that in LDM protein supplementation does not further enhance RT-induced muscle fiber hypertrophy nor influence mechanic muscle fiber characteristics but partially counteracts the fast-to-slow fiber shift.
doi:10.3390/nu8060331
PMCID: PMC4924172  PMID: 27258300
nutrition; supplementation; whey protein; myosin isoform; strength training; single muscle fiber mechanics
6.  Static and Dynamic Postural Changes after a Mountain Ultra-Marathon of 80 km and 5500 D+ 
PLoS ONE  2016;11(5):e0155085.
The study aimed to investigate the effect of fatigue on static and dynamic postural stability after completing a mountain ultra-marathon. Twelve male athletes participated in the study. Postural stability was assessed before and immediately after the race. Static postural stability was evaluated on a dynamometric platform with eyes opened (OE) and closed (CE). Dynamic postural stability was assessed with OE on an instrumented plate which allowed medio-lateral oscillations. Stabilometric data were affected by fatigue in the OE condition, concerning sway path velocity (p = 0.0006), sway area velocity (p = 0.0006), area of the confidence ellipse (p = 0.0016), maximal anterior-posterior (AP) (p = 0.0017) and medio-lateral (ML) (p = 0.0039) oscillations. In the CE condition the sway path velocity (p = 0.0334), the maximal ML oscillations (p = 0.0161) and the area of the confident ellipse (p = 0.0180) were also negatively influenced. Stabilogram diffusion analysis showed in the OE condition an increase of short-term diffusion coefficients considering the anterior-posterior direction (Dfys; p = 0.0023) and the combination of the two (Dfr2s; p = 0.0032). Equally, long term diffusion coefficients increased considering the anterior-posterior direction (Dfyl; p = 0.0093) and the combination of the two (Dfr2l; p = 0.0086). In CE condition greater values were detected for medio-lateral direction (Dfxl; p = 0.033), anterior-posterior direction (Dfyl; p = 0.0459) and the combination of the two (Dfr2l; p = 0.0048). The dynamic postural stability test showed an increase of the time spent with the edges of the plate on the floor (p = 0.0152). Our results showed that mountain ultra-marathon altered static stability more than dynamic stability. An involvement of cognitive resources to monitor postural stability after fatiguing could be the explanation of the worsening in the automatic task (quiet standing) and of the positive compensation in the less automatic task (dynamic standing on the instrumented plate).
doi:10.1371/journal.pone.0155085
PMCID: PMC4861257  PMID: 27159563
7.  Single muscle fiber proteomics reveals unexpected mitochondrial specialization 
EMBO Reports  2015;16(3):387-395.
Mammalian skeletal muscles are composed of multinucleated cells termed slow or fast fibers according to their contractile and metabolic properties. Here, we developed a high-sensitivity workflow to characterize the proteome of single fibers. Analysis of segments of the same fiber by traditional and unbiased proteomics methods yielded the same subtype assignment. We discovered novel subtype-specific features, most prominently mitochondrial specialization of fiber types in substrate utilization. The fiber type-resolved proteomes can be applied to a variety of physiological and pathological conditions and illustrate the utility of single cell type analysis for dissecting proteomic heterogeneity.
doi:10.15252/embr.201439757
PMCID: PMC4364878  PMID: 25643707
exercise; metabolism; mitochondria; muscle fibers; single cell
8.  Cellular aging of skeletal muscle: telomeric and free radical evidence that physical inactivity is responsible and not age 
Telomeres play an essential role in maintaining chromosomal integrity in the face of physiological stressors. Although the age-related shortening of TL (telomere length) in highly proliferative tissue is predominantly due to the replication process, the mechanism for telomere shortening in skeletal muscle, which is minimally proliferative, is unclear. By studying TL in both the upper and lower limbs of the young, old-mobile and old-immobile subjects and by virtue of the bipedal nature of human locomotion, which declines with age, it may be possible to elucidate the mechanism(s) responsible for cellular aging of skeletal muscle. With this approach, we revealed that TL (~15 kb) in arm skeletal muscle is unaffected by age. In contrast TL fell progressively in the legs across the young (~15 kb), the old mobile (~13 kb) and old immobile (~11 kb) subjects. Interestingly, there was a reciprocal increase in leg muscle free radicals across these groups that was correlated with TL (r = 0.7), with no such relationship in the arm (r = 0.09). Our results document that chronological age does not affect the cellular aging of skeletal muscle, but reveals that physical inactivity, probably mediated by free radicals, has a profound effect upon this process.
doi:10.1042/CS20140051
PMCID: PMC4757470  PMID: 24708050
aging; free radical; physical activity; telomere length; sarcopenia
9.  Protein Supplementation Increases Postexercise Plasma Myostatin Concentration After 8 Weeks of Resistance Training in Young Physically Active Subjects 
Journal of Medicinal Food  2015;18(1):137-143.
Abstract
Myostatin (MSTN) is a negative regulator of muscle growth even if some studies have shown a counterintuitive positive correlation between MSTN and muscle mass (MM). Our aim was to investigate the influence of 2 months of resistance training (RT) and diets with different protein contents on plasma MSTN, interleukin 1 beta (IL-1β), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), and insulin-like growth factor 1 (IGF-1). Eighteen healthy volunteers were randomly divided in two groups: high protein (HP) and normal protein (NP) groups. Different protein diet contents were 1.8 and 0.85 g of protein·kg bw−1·day−1 for HP and NP, respectively. Subjects underwent 8 weeks of standardized progressive RT. MSTN, IGF-1, IL-1β, IL-6, and TNF-α were analyzed before and after the first and the last training sessions. Lean body mass, MM, upper-limb muscle area, and strength were measured. Plasma MSTN showed a significant increase (P<.001) after the last training in the HP group compared with NP group and with starting value. IGF-1 plasma concentration showed a positive correlation with MSTN in HP after the last training (r2=0.6456; P=.0295). No significant differences were found between NP and HP for IL-1β, IL-6, TNF-α, and strength and MM or area. These findings suggest a “paradoxical” postexercise increase of plasma MSTN after 8 weeks of RT and HP diets. This MSTN elevation correlates positively with IGF-1 plasma level. This double increase of opposite (catabolic/anabolic) mediators could explain the substantial overlapping of MM increases in the two groups.
doi:10.1089/jmf.2014.0004
PMCID: PMC4281872  PMID: 25133710
cytokines; diet; exercise; myostatin; nutritional supplement
10.  A mutation in the CASQ1 gene causes a vacuolar myopathy with accumulation of sarcoplasmic reticulum protein aggregates 
Human mutation  2014;35(10):1163-1170.
A missense mutation in the calsequestrin-1 gene (CASQ1) was found in a group of patients with a myopathy characterized by weakness, fatigue and the presence of large vacuoles containing characteristic inclusions resulting from the aggregation of sarcoplasmic reticulum (SR) proteins. The mutation affects a conserved aspartic acid in position 244 (p.Asp244Gly) located in one of the high-affinity Ca2+ binding sites of CASQ1 and alters the kinetics of Ca2+ release in muscle fibers. Expression of the mutated CASQ1 protein in COS-7 cells showed a markedly reduced ability in forming elongated polymers, while both in cultured myotubes and in in-vivo mouse fibers induced the formation of electron-dense SR vacuoles containing aggregates of the mutant CASQ1 protein that resemble those observed in muscle biopsies of patients. Altogether, these results support the view that a single missense mutation in the CASQ1 gene causes the formation of abnormal SR vacuoles containing aggregates of CASQ1 and other SR proteins, results in altered Ca2+ release in skeletal muscle fibers and, hence, is responsible for the clinical phenotype observed in these patients.
doi:10.1002/humu.22631
PMCID: PMC4177304  PMID: 25116801
aggregate myopathy; CASQ1; calsequestrin; sarcoplasmic reticulum; skeletal muscle
11.  Biomechanical Comparison of Shorts With Different Pads 
Medicine  2015;94(29):e1186.
Abstract
An intensive use of the bicycle may increase the risk of erectile dysfunction and the compression of the perineal area has been showed to be a major mechanism leading to sexual alterations compromising the quality of life. Manufacturers claim that pads contribute to increase cyclists perineal protection ensuring a high level of comfort.
To investigate the influence of various cycling pads with regard to perineal protection and level of comfort.
Nine club road cyclists rode 20 min on a drum simulator, located at the Nutrition and Exercise Physiology Laboratory, at a constant speed and gear ratio wearing the shorts with 3 cycling pads of different design and thickness: basic (BAS), intermediate (INT), and endurance (END). Kinematics and pressure data were recorded at min 5, 15, and 20 of the test using a motion capture system and a pressure sensor mat. The variables of interest were: 3-dimensional pelvis excursions, peak pressure, mean pressure, and vertical force. The comfort level was assessed with a ranking order based on the subjects’ perception after the 20-min trials and measuring the vertical ground reaction force under the anterior wheel as well as the length of the center of pressure (COP) trajectory on the saddle.
Results showed that the vertical force and the average value of mean pressure on the saddle significantly decreased during the 20-min period of testing for BAS and END. Mean peak pressure on the corresponding perineal cyclist area significantly increased only for BAS during the 20-min period. Interestingly objective comfort indexes measured did not match cyclists subjective comfort evaluation.
The lower capacity of BAS to reduce the peak pressure on the corresponding perineal area after 20 min of testing, together with its positive comfort evaluation, suggest that a balance between protection and perceived comfort should be taken into account in the choice of the pad. Hence, the quantitative approach of objective comfort indexes introduced in this study could be helpful for manufacturers in the development of their protective pads.
doi:10.1097/MD.0000000000001186
PMCID: PMC4603024  PMID: 26200626
12.  Developmental myosins: expression patterns and functional significance 
Skeletal Muscle  2015;5:22.
Developing skeletal muscles express unique myosin isoforms, including embryonic and neonatal myosin heavy chains, coded by the myosin heavy chain 3 (MYH3) and MYH8 genes, respectively, and myosin light chain 1 embryonic/atrial, encoded by the myosin light chain 4 (MYL4) gene. These myosin isoforms are transiently expressed during embryonic and fetal development and disappear shortly after birth when adult fast and slow myosins become prevalent. However, developmental myosins persist throughout adult stages in specialized muscles, such as the extraocular and jaw-closing muscles, and in the intrafusal fibers of the muscle spindles. These myosins are re-expressed during muscle regeneration and provide a specific marker of regenerating fibers in the pathologic skeletal muscle. Mutations in MYH3 or MYH8 are responsible for distal arthrogryposis syndromes, characterized by congenital joint contractures and orofacial dysmorphisms, supporting the importance of muscle contractile activity and body movements in joint development and in shaping the form of the face during fetal development. The biochemical and biophysical properties of developmental myosins have only partially been defined, and their functional significance is not yet clear. One possibility is that these myosins are specialized in contracting against low loads, and thus, they may be adapted to the prenatal environment, when fetal muscles contract against a very low load compared to postnatal muscles.
doi:10.1186/s13395-015-0046-6
PMCID: PMC4502549  PMID: 26180627
Muscle development; Muscle regeneration; Myosin heavy chain; Embryonic myosin; Neonatal myosin; Distal arthrogryposis
13.  Oxidative stress, mitochondrial damage, and cores in muscle from calsequestrin-1 knockout mice 
Skeletal Muscle  2015;5:10.
Background
Mutations in the gene encoding ryanodine receptor type-1 (RYR1), the calcium ion (Ca2+) release channel in the sarcoplasmic reticulum (SR) of skeletal muscle, are linked to central core disease (CCD) and malignant hyperthermia (MH) susceptibility. We recently reported that mice lacking the skeletal isoform of calsequestrin (CASQ1-null), the primary Ca2+ buffer in the SR of skeletal muscle and a modulator of RYR1 activity, exhibit lethal heat- and anesthetic-induced hypermetabolic episodes that resemble MH events in humans.
Methods
We compared ultrastructure, oxidative status, and contractile function in skeletal fibers of extensor digitorum longus (EDL) muscles in wild type (WT) and CASQ1-null mice at different ages (from 4 to 27 months) using structural, biochemical, and functional assays.
Results
About 25% of fibers in EDL muscles from CASQ1-null mice of 14 to 27 months of age exhibited large areas of structural disarray (named core-like regions), which were rarely observed in muscle from age-matched WT mice. To determine early events that may lead to the formation of cores, we analyzed EDL muscles from adult mice: at 4 to 6 months of age, CASQ1-null mice (compared to WT) displayed significantly reduced grip strength (40 ± 1 vs. 86 ± 1 mN/gr) and exhibited an increase in the percentage of damaged mitochondria (15.1% vs. 2.6%) and a decrease in average cross-sectional fiber area (approximately 37%) in EDL fibers. Finally, oxidative stress was also significantly increased (25% reduction in ratio between reduced and oxidized glutathione, or GSH/GSSG, and 35% increase in production of mitochondrial superoxide flashes). Providing ad libitum access to N-acetylcysteine in the drinking water for 2 months normalized GSH/GSSG ratio, reduced mitochondrial damage (down to 8.9%), and improved grip strength (from 46 ± 3 to 59 ± 2 mN/gr) in CASQ1-null mice.
Conclusions
Our findings: 1) demonstrate that ablation of CASQ1 leads to enhanced oxidative stress, mitochondrial damage, and the formation of structural cores in skeletal muscle; 2) provide new insights in the pathogenic mechanisms that lead to damage/disappearance of mitochondria in cores; and 3) suggest that antioxidants may provide some therapeutic benefit in reducing mitochondrial damage, limiting the development of cores, and improving muscle function.
Electronic supplementary material
The online version of this article (doi:10.1186/s13395-015-0035-9) contains supplementary material, which is available to authorized users.
doi:10.1186/s13395-015-0035-9
PMCID: PMC4464246  PMID: 26075051
Excitation-contraction (EC) coupling; Mitochondria; Ryanodine receptor (RYR)
14.  Single muscle fiber proteomics reveals unexpected mitochondrial specialization 
EMBO Reports  2015;16(3):387-395.
Mammalian skeletal muscles are composed of multinucleated cells termed slow or fast fibers according to their contractile and metabolic properties. Here, we developed a high-sensitivity workflow to characterize the proteome of single fibers. Analysis of segments of the same fiber by traditional and unbiased proteomics methods yielded the same subtype assignment. We discovered novel subtype-specific features, most prominently mitochondrial specialization of fiber types in substrate utilization. The fiber type-resolved proteomes can be applied to a variety of physiological and pathological conditions and illustrate the utility of single cell type analysis for dissecting proteomic heterogeneity.
doi:10.15252/embr.201439757
PMCID: PMC4364878  PMID: 25643707
exercise; metabolism; mitochondria; muscle fibers; single cell
15.  Mitochondrial Ca2+-Handling in Fast Skeletal Muscle Fibers from Wild Type and Calsequestrin-Null Mice 
PLoS ONE  2013;8(10):e74919.
Mitochondrial calcium handling and its relation with calcium released from sarcoplasmic reticulum (SR) in muscle tissue are subject of lively debate. In this study we aimed to clarify how the SR determines mitochondrial calcium handling using dCASQ-null mice which lack both isoforms of the major Ca2+-binding protein inside SR, calsequestrin. Mitochondrial free Ca2+-concentration ([Ca2+]mito) was determined by means of a genetically targeted ratiometric FRET-based probe. Electron microscopy revealed a highly significant increase in intermyofibrillar mitochondria (+55%) and augmented coupling (+12%) between Ca2+ release units of the SR and mitochondria in dCASQ-null vs. WT fibers. Significant differences in the baseline [Ca2+]mito were observed between quiescent WT and dCASQ-null fibers, but not in the resting cytosolic Ca2+ concentration. The rise in [Ca2+]mito during electrical stimulation occurred in 20−30 ms, while the decline during and after stimulation was governed by 4 rate constants of approximately 40, 1.6, 0.2 and 0.03 s−1. Accordingly, frequency-dependent increase in [Ca2+]mito occurred during sustained contractions. In dCASQ-null fibers the increases in [Ca2+]mito were less pronounced than in WT fibers and even lower when extracellular calcium was removed. The amplitude and duration of [Ca2+]mito transients were increased by inhibition of mitochondrial Na+/Ca2+ exchanger (mNCX). These results provide direct evidence for fast Ca2+ accumulation inside the mitochondria, involvement of the mNCX in mitochondrial Ca2+-handling and a dependence of mitochondrial Ca2+-handling on intracellular (SR) and external Ca2+ stores in fast skeletal muscle fibers. dCASQ-null mice represent a model for malignant hyperthermia. The differences in structure and in mitochondrial function observed relative to WT may represent compensatory mechanisms for the disease-related reduction of calcium storage capacity of the SR and/or SR Ca2+-leakage.
doi:10.1371/journal.pone.0074919
PMCID: PMC3789688  PMID: 24098358
16.  Obscurin is required for ankyrinB-dependent dystrophin localization and sarcolemma integrity 
The Journal of Cell Biology  2013;200(4):523-536.
Obscurin contributes to the organization of subsarcolemma microtubules, localization of dystrophin at costameres, and maintenance of sarcolemmal integrity in skeletal muscle fibers.
Obscurin is a large myofibrillar protein that contains several interacting modules, one of which mediates binding to muscle-specific ankyrins. Interaction between obscurin and the muscle-specific ankyrin sAnk1.5 regulates the organization of the sarcoplasmic reticulum in striated muscles. Additional muscle-specific ankyrin isoforms, ankB and ankG, are localized at the subsarcolemma level, at which they contribute to the organization of dystrophin and β-dystroglycan at costameres. In this paper, we report that in mice deficient for obscurin, ankB was displaced from its localization at the M band, whereas localization of ankG at the Z disk was not affected. In obscurin KO mice, localization at costameres of dystrophin, but not of β-dystroglycan, was altered, and the subsarcolemma microtubule cytoskeleton was disrupted. In addition, these mutant mice displayed marked sarcolemmal fragility and reduced muscle exercise tolerance. Altogether, the results support a model in which obscurin, by targeting ankB at the M band, contributes to the organization of subsarcolemma microtubules, localization of dystrophin at costameres, and maintenance of sarcolemmal integrity.
doi:10.1083/jcb.201205118
PMCID: PMC3575540  PMID: 23420875
17.  Myosin Isoforms and Contractile Properties of Single Fibers of Human Latissimus Dorsi Muscle 
BioMed Research International  2013;2013:249398.
The aim of our study was to investigate fiber type distribution and contractile characteristics of Latissimus Dorsi muscle (LDM). Samples were collected from 18 young healthy subjects (9 males and 9 females) through percutaneous fine needle muscle biopsy. The results showed a predominance of fast myosin heavy chain isoforms (MyHC) with 42% of MyHC 2A and 25% of MyHC 2X, while MyHC 1 represented only 33%. The unbalance toward fast isoforms was even greater in males (71%) than in females (64%). Fiber type distribution partially reflected MyHC isoform distribution with 28% type 1/slow fibers and 5% hybrid 1/2A fibers, while fast fibers were divided into 30% type 2A, 31% type A/X, 4% type X, and 2% type 1/2X. Type 1/slow fibers were not only less abundant but also smaller in cross-sectional area than fast fibers. During maximal isometric contraction, type 1/slow fibers developed force and tension significantly lower than the two major groups of fast fibers. In conclusion, the predominance of fast fibers and their greater size and strength compared to slow fibers reveal that LDM is a muscle specialized mainly in phasic and powerful activity. Importantly, such specialization is more pronounced in males than in females.
doi:10.1155/2013/249398
PMCID: PMC3736486  PMID: 23971027
18.  Correction: AQP4-Dependent Water Transport Plays a Functional Role in Exercise-Induced Skeletal Muscle Adaptations 
PLoS ONE  2013;8(6):10.1371/annotation/86fc2632-913c-490d-8b9b-e925b38baec5.
doi:10.1371/annotation/86fc2632-913c-490d-8b9b-e925b38baec5
PMCID: PMC3731368
19.  AQP4-Dependent Water Transport Plays a Functional Role in Exercise-Induced Skeletal Muscle Adaptations 
PLoS ONE  2013;8(3):e58712.
In this study we assess the functional role of Aquaporin-4 (AQP4) in the skeletal muscle by analyzing whether physical activity modulates AQP4 expression and whether the absence of AQP4 has an effect on osmotic behavior, muscle contractile properties, and physical activity. To this purpose, rats and mice were trained on the treadmill for 10 (D10) and 30 (D30) days and tested with exercise to exhaustion, and muscles were used for immunoblotting, RT-PCR, and fiber-type distribution analysis. Taking advantage of the AQP4 KO murine model, functional analysis of AQP4 was performed on dissected muscle fibers and sarcolemma vesicles. Moreover, WT and AQP4 KO mice were subjected to both voluntary and forced activity. Rat fast-twitch muscles showed a twofold increase in AQP4 protein in D10 and D30 rats compared to sedentary rats. Such increase positively correlated with the animal performance, since highest level of AQP4 protein was found in high runner rats. Interestingly, no shift in muscle fiber composition nor an increase in AQP4-positive fibers was found. Furthermore, no changes in AQP4 mRNA after exercise were detected, suggesting that post-translational events are likely to be responsible for AQP4 modulation. Experiments performed on AQP4 KO mice revealed a strong impairment in osmotic responses as well as in forced and voluntary activities compared to WT mice, even though force development amplitude and contractile properties were unvaried. Our findings definitively demonstrate the physiological role of AQP4 in supporting muscle contractile activity and metabolic changes that occur in fast-twitch skeletal muscle during prolonged exercise.
doi:10.1371/journal.pone.0058712
PMCID: PMC3592820  PMID: 23520529
20.  Differential Effect of Calsequestrin Ablation on Structure and Function of Fast and Slow Skeletal Muscle Fibers 
We compared structure and function of EDL and Soleus muscles in adult (4–6 m) mice lacking both Calsequestrin (CASQ) isoforms, the main SR Ca2+-binding proteins. Lack of CASQ induced ultrastructural alterations in ~30% of Soleus fibers, but not in EDL. Twitch time parameters were prolonged in both muscles, although tension was not reduced. However, when stimulated for 2 sec at 100 hz, Soleus was able to sustain contraction, while in EDL active tension declined by 70–80%. The results presented in this paper unmask a differential effect of CASQ1&2 ablation in fast versus slow fibers. CASQ is essential in EDL to provide large amount of Ca2+ released from the SR during tetanic stimulation. In contrast, Soleus deals much better with lack of CASQ because slow fibers require lower Ca2+ amounts and slower cycling to function properly. Nevertheless, Soleus suffers more severe structural damage, possibly because SR Ca2+ leak is more pronounced.
doi:10.1155/2011/634075
PMCID: PMC3173739  PMID: 21941434
21.  Microgenomic Analysis in Skeletal Muscle: Expression Signatures of Individual Fast and Slow Myofibers 
PLoS ONE  2011;6(2):e16807.
Background
Skeletal muscle is a complex, versatile tissue composed of a variety of functionally diverse fiber types. Although the biochemical, structural and functional properties of myofibers have been the subject of intense investigation for the last decades, understanding molecular processes regulating fiber type diversity is still complicated by the heterogeneity of cell types present in the whole muscle organ.
Methodology/Principal Findings
We have produced a first catalogue of genes expressed in mouse slow-oxidative (type 1) and fast-glycolytic (type 2B) fibers through transcriptome analysis at the single fiber level (microgenomics). Individual fibers were obtained from murine soleus and EDL muscles and initially classified by myosin heavy chain isoform content. Gene expression profiling on high density DNA oligonucleotide microarrays showed that both qualitative and quantitative improvements were achieved, compared to results with standard muscle homogenate. First, myofiber profiles were virtually free from non-muscle transcriptional activity. Second, thousands of muscle-specific genes were identified, leading to a better definition of gene signatures in the two fiber types as well as the detection of metabolic and signaling pathways that are differentially activated in specific fiber types. Several regulatory proteins showed preferential expression in slow myofibers. Discriminant analysis revealed novel genes that could be useful for fiber type functional classification.
Conclusions/Significance
As gene expression analyses at the single fiber level significantly increased the resolution power, this innovative approach would allow a better understanding of the adaptive transcriptomic transitions occurring in myofibers under physiological and pathological conditions.
doi:10.1371/journal.pone.0016807
PMCID: PMC3043066  PMID: 21364935
22.  Two novel/ancient myosins in mammalian skeletal muscles: MYH14/7b and MYH15 are expressed in extraocular muscles and muscle spindles 
The Journal of Physiology  2009;588(Pt 2):353-364.
The mammalian genome contains three ancient sarcomeric myosin heavy chain (MYH) genes, MYH14/7b, MYH15 and MYH16, in addition to the two well characterized clusters of skeletal and cardiac MYHs. MYH16 is expressed in jaw muscles of carnivores; however the expression pattern of MYH14 and MYH15 is not known. MYH14 and MYH15 orthologues are present in frogs and birds, coding for chicken slow myosin 2 and ventricular MYH, respectively, whereas only MYH14 orthologues have been detected in fish. In all species the MYH14 gene contains a microRNA, miR-499. Here we report that in rat and mouse, MYH14 and miR-499 transcripts are detected in heart, slow muscles and extraocular (EO) muscles, whereas MYH15 transcripts are detected exclusively in EO muscles. However, MYH14 protein is detected only in a minor fibre population in EO muscles, corresponding to slow-tonic fibres, and in bag fibres of muscle spindles. MYH15 protein is present in most fibres of the orbital layer of EO muscles and in the extracapsular region of bag fibres. During development, MYH14 is expressed at low levels in skeletal muscles, heart and all EO muscle fibres but disappears from most fibres, except the slow-tonic fibres, after birth. In contrast, MYH15 is absent in embryonic and fetal muscles and is first detected after birth in the orbital layer of EO muscles. The identification of the expression pattern of MYH14 and MYH15 brings to completion the inventory of the MYH isoforms involved in sarcomeric architecture of skeletal muscles and provides an unambiguous molecular basis to study the contractile properties of slow-tonic fibres in mammals.
doi:10.1113/jphysiol.2009.181008
PMCID: PMC2821527  PMID: 19948655
23.  Muscle Research and Gene Ontology: New standards for improved data integration 
Background
The Gene Ontology Project provides structured controlled vocabularies for molecular biology that can be used for the functional annotation of genes and gene products. In a collaboration between the Gene Ontology (GO) Consortium and the muscle biology community, we have made large-scale additions to the GO biological process and cellular component ontologies. The main focus of this ontology development work concerns skeletal muscle, with specific consideration given to the processes of muscle contraction, plasticity, development, and regeneration, and to the sarcomere and membrane-delimited compartments. Our aims were to update the existing structure to reflect current knowledge, and to resolve, in an accommodating manner, the ambiguity in the language used by the community.
Results
The updated muscle terminologies have been incorporated into the GO. There are now 159 new terms covering critical research areas, and 57 existing terms have been improved and reorganized to follow their usage in muscle literature.
Conclusion
The revised GO structure should improve the interpretation of data from high-throughput (e.g. microarray and proteomic) experiments in the area of muscle science and muscle disease. We actively encourage community feedback on, and gene product annotation with these new terms. Please visit the Muscle Community Annotation Wiki .
doi:10.1186/1755-8794-2-6
PMCID: PMC2657163  PMID: 19178689
24.  Two novel/ancient myosins in mammalian skeletal muscles: MYH14/7b and MYH15 are expressed in extraocular muscles and muscle spindles 
The Journal of Physiology  2009;588(2):353-364.
The mammalian genome contains three ancient sarcomeric myosin heavy chain (MYH) genes, MYH14/7b, MYH15 and MYH16, in addition to the two well characterized clusters of skeletal and cardiac MYHs. MYH16 is expressed in jaw muscles of carnivores; however the expression pattern of MYH14 and MYH15 is not known. MYH14 and MYH15 orthologues are present in frogs and birds, coding for chicken slow myosin 2 and ventricular MYH, respectively, whereas only MYH14 orthologues have been detected in fish. In all species the MYH14 gene contains a microRNA, miR-499. Here we report that in rat and mouse, MYH14 and miR-499 transcripts are detected in heart, slow muscles and extraocular (EO) muscles, whereas MYH15 transcripts are detected exclusively in EO muscles. However, MYH14 protein is detected only in a minor fibre population in EO muscles, corresponding to slow-tonic fibres, and in bag fibres of muscle spindles. MYH15 protein is present in most fibres of the orbital layer of EO muscles and in the extracapsular region of bag fibres. During development, MYH14 is expressed at low levels in skeletal muscles, heart and all EO muscle fibres but disappears from most fibres, except the slow-tonic fibres, after birth. In contrast, MYH15 is absent in embryonic and fetal muscles and is first detected after birth in the orbital layer of EO muscles. The identification of the expression pattern of MYH14 and MYH15 brings to completion the inventory of the MYH isoforms involved in sarcomeric architecture of skeletal muscles and provides an unambiguous molecular basis to study the contractile properties of slow-tonic fibres in mammals.
doi:10.1113/jphysiol.2009.181008
PMCID: PMC2821527  PMID: 19948655

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