WT Fibers Research Articles

Early Developmental Changes of Muscle Acetylcholine Receptors Are Little Influenced by Dystrophin Absence in mdx Mouse.

Dystrophin is a cytoskeletal protein contributing to the organization of the neuromuscular junction. In Duchenne muscular dystrophy, due to dystrophin absence, the distribution of endplate acetylcholine receptors (AChRs) becomes disorganized. It is still debated whether this is due to the absence of dystrophin or to the repeated damage/regeneration cycles typical of dystrophic muscle. We addressed this controversy studying the endplate in the first 3 postnatal weeks, when muscle damage in dystrophic (mdx) mice is minimal. By synaptic and extra-synaptic patch-clamp recordings in acutely dissociated mdx and wt muscle fibers, we recorded unitary events due to openings of AChR-channels containing the γ and ε subunit. We also examined AChR distribution at the endplate by immunofluorescence assays. No differences between wt and mdx fibers were found in the γ/ε switch, nor in the AChR organization at the endplates up to 21 postnatal days. Conversely, we detected a delayed appearance and disappearance of patches with high channel opening frequency in mdx fibers. Our data emphasize that the innervation-dependent γ/ε switch and AChR organization in the endplate are not affected by the absence of dystrophin, while extra-synaptic AChR cluster formation and disassembly could be differentially regulated in mdx mice.

ANO5 ensures trafficking of annexins in wounded myofibers.

Mutations in ANO5 (TMEM16E) cause limb-girdle muscular dystrophy R12. Defective plasma membrane repair is a likely mechanism. Using myofibers from Ano5 knockout mice, we show that trafficking of several annexin proteins, which together form a cap at the site of injury, is altered upon loss of ANO5. Annexin A2 accumulates at the wound to nearly twice the level observed in WT fibers, while annexin A6 accumulation is substantially inhibited in the absence of ANO5. Appearance of annexins A1 and A5 at the cap is likewise diminished in the Ano5 knockout. These changes are correlated with an alteration in annexin repair cap fine structure and shedding of annexin-positive vesicles. We conclude that loss of annexin coordination during repair is disrupted in Ano5 knockout mice and underlies the defective repair phenotype. Although ANO5 is a phospholipid scramblase, abnormal repair is rescued by overexpression of a scramblase-defective ANO5 mutant, suggesting a novel, scramblase-independent role of ANO5 in repair.

Pathophysiological Consequences of KATP Channel Overactivity and Pharmacological Response to Glibenclamide in Skeletal Muscle of a Murine Model of Cantù Syndrome.

Cantù syndrome (CS) arises from mutations in ABCC9 and KCNJ8 genes that lead to gain of function (GOF) of ATP-sensitive potassium (KATP) channels containing SUR2A and Kir6.1 subunits, respectively, of KATP channels. Pathological consequences of CS have been reported for cardiac and smooth muscle cells but consequences in skeletal muscle are unknown. Children with CS show muscle hypotonia and adult manifest fatigability. We analyzed muscle properties of Kir6.1[V65M] CS mice, by measurements of forelimb strength and ultrasonography of hind-limb muscles, as well as assessing KATP channel properties in native Flexor digitorum brevis (FDB) and Soleus (SOL) fibers by the patch-clamp technique in parallel with histopathological, immunohistochemical and Polymerase Chain Reaction (PCR) analysis. Forelimb strength was lower in Kir6.1wt/VM mice than in WT mice. Also, a significant enhancement of echodensity was observed in hind-limb muscles of Kir6.1wt/VM mice relative to WT, suggesting the presence of fibrous tissue. There was a higher KATP channel current amplitude in Kir6.1wt/VM FDB fibers relative to WT and a reduced response to glibenclamide. The IC50 of glibenclamide to block KATP channels in FDB fibers was 1.3 ± 0.2 × 10−7 M in WT and 1.2 ± 0.1 × 10−6 M in Kir6.1wt/VM mice, respectively; and it was 1.2 ± 0.4 × 10−7 M in SOL WT fibers but not measurable in Kir6.1wt/VM fibers. The sensitivity of the KATP channel to MgATP was not modified in Kir6.1wt/VM fibers. Histopathological/immunohistochemical analysis of SOL revealed degeneration plus regressive-necrotic lesions with regeneration, and up-regulation of Atrogin-1, MuRF1, and BNIP3 mRNA/proteins in Kir6.1wt/VM mice. Kir6.1wt/VM mutation in skeletal muscle leads to changes of the KATP channel response to glibenclamide in FDB and SOL fibers, and it is associated with histopathological and gene expression changes in slow-twitch muscle, suggesting marked atrophy and autophagy.

Sarcolemma wounding activates dynamin-dependent endocytosis in striated muscle.

Plasma membrane repair is an evolutionarily conserved mechanism by which cells can seal breaches in the plasma membrane. Mutations in several proteins with putative roles in sarcolemma integrity, membrane repair, and membrane transport result in several forms of muscle disease; however, the mechanisms that are activated and responsible for sarcolemma resealing are not well understood. Using the standard assays for membrane repair, which track the uptake of FM 1-43 dye into adult skeletal muscle fibers following laser-induced sarcolemma disruption, we show that labeling of resting fibers by FM1-43 prior to membrane wounding and the induced FM1-43 dye uptake after sarcolemma wounding occurs via dynamin-dependent endocytosis. Dysferlin-deficient muscle fibers show elevated dye uptake following wounding, which is the basis for the assertion that membrane repair is defective in this model. Our data show that dynamin inhibition mitigates the differences in FM1-43 dye uptake between dysferlin-null and wild-type muscle fibers, suggesting that elevated wound-induced FM1-43 uptake in dysferlin-deficient muscle may actually be due to enhanced dynamin-dependent endocytosis following wounding, though dynamin inhibition had no effect on dysferlin trafficking after wounding. By monitoring calcium flux after membrane wounding, we show that reversal of calcium precedes the sustained, slower increase of dynamin-dependent FM1-43 uptake in WT fibers, and that dysferlin-deficient muscle fibers have persistently increased calcium after wounding, consistent with its proposed role in resealing. These data highlight a previously unappreciated role for dynamin-dependent endocytosis in wounded skeletal muscle fibers and identify overactive dynamin-dependent endocytosis following sarcolemma wounding as a potential mechanism or consequence of dysferlin deficiency.

Post-Developmental Knockout of Orai1-Mediated Store-Operated Calcium Entry Improves Muscle Pathology in a Mouse Model of Muscular Dystrophy

Duchenne muscular dystrophy (DMD), a genetic disorder caused by mutations in the dystrophin gene, is characterized by progressive muscle degeneration/weakness. Enhanced store-operated Ca2+ entry (SOCE), a Ca2+ influx mechanism coordinated by STIM1 and Orai1, is proposed to potentiate Ca2+-mediated damage in DMD. To determine the role of SOCE in promoting muscular dystrophy, we crossed mdx mice with tamoxifen-inducible, muscle-specific Orai1 knockout mice (mdx-Orai1 KO). Analysis of transcript levels revealed that Orai1 expression was ∼1.5x higher in muscle from mdx mice compared to WT mice, while nearly undetectable in muscle from mdx-Orai1 KO mice. Levels of Orai1 transcript correlated with SOCE activity measured using Mn2+ quench of fura-2 fluorescence in single flexor digitorum brevis muscle fibers. Specifically, SOCE was increased in mdx fibers and absent in fibers from mdx-Orai1 KO mice. Moreover, compared to WT fibers, mdx fibers exhibited: i) increased myoplasmic Ca2+ levels; ii) reduced Ca2+ store content; and iii) a significant prolongation in the rate of electrically-evoked Ca2+ transient decay. All of these effects were normalized in fibers from mdx-Orai1 KO mice. Ex vivo contractility experiments revealed that extensor digitorum longus muscles from mdx mice displayed a reduction in maximal tetanic specific force (150.3±3.5 mN/mm2) compared to WT muscles (199.6±11.1 mN/mm2), a parameter ameliorated in muscles from mdx-Orai1 KO mice (188.1±10.9 mN/mm2). When exposed to 10 consecutive eccentric contractions, mdx muscles exhibited a higher damage-induced force reduction than WT muscles (residual force after the 10th eccentric contraction was 35.5±3.8% and 61.8±1.9%, respectively). Interestingly, Orai1 ablation partially protected muscles from damage in mdx-Orai1 KO mice (residual force was 46.1±2.6% after 10 contractions). Our results indicate that enhanced Orai1-dependent SOCE exacerbates the dystrophic phenotype and Orai1 deficiency promotes sarcolemmal integrity/stability during eccentric contraction.

Mechanical and thermal conductivity properties of hemp fiber reinforced polyurethane composites

The aim of the present work is to introduce natural hemp fiber as reinforcement in the preparation of partially biodegradable green composites. Composite of rigid polyurethane (PU) and hemp fiber (H.F) ​​were prepared at different loading rates in (H.F) ​​(5%, 10%, 15%, 20%, 25% and 30%). Water absorption, thermal conductivity, and mechanical properties of composite were investigated as a function of fiber content. The results show that, the thermal conductivity of composites increases linearly with density. The mechanical properties of composites with 15% wt fibers loading provided a 40% increase in strength. Measured properties showed that polyurethane- hemp fibers composite present good insulating properties compared to the traditional insulation materials (glass wool, mineral wool etc.). Therefore, (PU-HF) insulation may provide a promising solution for building insulation.

Distinct Lattice Structure Altreations in DCM and HCM Mouse Models Associated with Mutations in Myosin Regulatory Light Chain

In this study we used small angle X-ray diffraction to analyze changes in the interfilament lattice spacing (IFS) and equatorial reflections’ intensity ratio (I1,1/I1,0) due to mutations in myosin RLC shown to cause hypertrophic (HCM) or dilated (DCM) cardiomyopathy. In mice, D166V was found to result in systolic and diastolic dysfunction, reduced contractile force and abnormally high Ca2+-sensitivity, highlighting the severity of the HCM phenotype. The D94A mutation resulted in DCM manifested by increased LV-chamber diameter and reduced ejection fraction with no changes in maximal force production but with D94A-induced decrease in the Ca2+-sensitivity of force. In this report we collected the X-ray data on skinned papillary muscles from D166V-HCM and D94A-DCM vs. WT mice at serial pCa (8, 7, 6, 5.4, 5.2, and 4) and at SL=2.1µm. For D166V-HCM vs. WT-fibers, IFS was significantly increased in pCa8, 6 and 5.4 but significantly decreased at pCa4. For D94A-DCM and for WT, no significant changes in IFS were observed within or between the groups for each pCa. There was a clear correlation between I1,1/I1,0 readings vs. increasing [Ca2+] for all three groups of fibers. A significant increase in I1,1/I1,0 for all pCa solutions (except pCa4) was observed in D166V-HCM vs. WT fibers. These data correlated with D166V-induced changes in force/Ca2+-sensitivity observed in fiber mechanics study. For D94A-DCM, a near significant decrease in I1,1/I1,0 at pCa 5.2 vs. WT was observed (WT:0.43±0.08 vs. D94A: 0.35±0.09 P=0.072). Fitting I1,1/I1,0 –pCa plots yielded pCa50=5.2±0.03 (WT), 4.98±0.05 (D94A) and 5.43±0.10 (D166V), showing a clear correlation in pCa50-values between X-ray and fiber mechanics experiments. Our collective data indicate that RLC mutations lead to HCM or DCM through structural rearrangements of myosin cross-bridge mass with D166V bringing it closer to actin, and D94A moving it farther away from the thin-filaments. Supported by NIH-HL123255 (DSC).

Tip‐Enhanced Raman Spectroscopy to Distinguish Toxic Oligomers from Aβ1–42 Fibrils at the Nanometer Scale

AbstractFor the first time, natural Aβ1–42 fibrils (WT) implicated in Alzheimer's disease, as well as two synthetic mutants forming less toxic amyloid fibrils (L34T) and highly toxic oligomers (oG37C), are chemically characterized at the scale of a single structure using tip‐enhanced Raman spectroscopy (TERS). While the proportion of TERS features associated with amino acid residues is similar for the three peptides, a careful examination of amide I and amide III bands allows us to clearly distinguish WT and L34T fibers organized in parallel β‐sheets from the small and more toxic oG37C oligomers organized in anti‐parallel β‐sheets.

Tip-Enhanced Raman Spectroscopy to Distinguish Toxic Oligomers from Aβ1-42 Fibrils at the Nanometer Scale.

For the first time, natural Aβ1-42 fibrils (WT) implicated in Alzheimer's disease, as well as two synthetic mutants forming less toxic amyloid fibrils (L34T) and highly toxic oligomers (oG37C), are chemically characterized at the scale of a single structure using tip-enhanced Raman spectroscopy (TERS). While the proportion of TERS features associated with amino acid residues is similar for the three peptides, a careful examination of amide I and amide III bands allows us to clearly distinguish WT and L34T fibers organized in parallel β-sheets from the small and more toxic oG37C oligomers organized in anti-parallel β-sheets.

Effect of Mutations in cMyBP-C on Sarcomere Mechanical Function

A significant number of cardiac myopathies in children and adults are caused by mutations in the cMyBP-C gene. In disease, cardiac output is compromised by altered cardiac muscle fiber contractility due to modulated interactions between cMyBP with actin or with S2. Comparative measurements in WT and knockout mice (cMyBP-C-/-) muscle fibers have showed increased isotonic shortening velocity, power output and rate of force redevelopment in absence cMyBP-C. Thus, change of only a few amino acids in mutant cMyBP-C, especially in regions rich with phosphorylation sites, may cause significant change in dynamics of muscle contractility. Comparison of measured sliding velocities of actin filaments over the regions of myosin filament with and without cMyBP-C in motility assays have provided molecular insight how these structural changes alter the kinetics of the interactions of cMyBP-C with myosin and actin filaments. We used a multi-scale, computational modelling platform, MUSICO, (MUscle SImulation COde) to assess the effect of cMyBP-C mutations on sarcomere contraction. This platform includes explicit 3-D sarcomere structures, extensible actin and myosin filaments, various models for the actomyosin cycles, thin filament regulation via a continuous flexible chain (CFC) model and now cMyBP-C using the kinetic parameters for dynamically forming and disrupting connections between cMyBP-C and actin, derived from the motility studies. We compared the model predictions between different mutations and the corresponding mechanical experiments. The predictions from cMyBP-C sarcomeric model showed significant differences between the mutants, and closely followed observations. This results allow the quantitative evaluation of the role of cMyBP-C in the regulation of sarcomere structure and function, the development of a multi-scale myoarchitectural representation of disease phenotype and the creation of a novel diagnostic and prognostic methodology for tracking disease progression in patients.Supported by: R01s AR048776 and DC 011528

Influence of Glass Fiber wt% and Silanization on Mechanical Flexural Strength of Reinforced Acrylics

The aim is to evaluate the flexural strength of acrylic resin bars depending on the addiction of glass fibers with or without previous 3-methacryloxypropyl-trimethoxysilane (silane) application. Short fibers (3 mm) were treated and added to an acrylic resin powder, being further mixed with acrylic liquid to create bars (25 × 2 × 2 mm) of 11 experimental groups (N = 10), according to the interaction of experimental factors: weight % of glass fibers: (0.5; 1; 3; 4; 6 and 7) and silane application (with silane (S) or without silane (N)). Flexural strength and scanning microscopy evaluation were performed (SEM). Data (MPa) were submitted to ANOVA and Tukey (α = 5%). A significant difference between groups was observed (p = 0.001): S7%(128.85 ± 35.76)a, S6% (119.31 ± 11.97)ab, S4% (116.98 ± 25.23)ab, N4% (107.85 ± 24.88)abc, S1% (96.29 ± 20.65)bc, S0.5% (89.29 ± 7.33)cd, S3% (89.0 ± 11.27)cd, N3% (86.79 ± 17.63)cd, N1% (85.43 ± 16.44)cd, Control (73.29 ± 25.0)de, N0.5% (59.58 ± 19.46)e. For N groups, it was not possible to include more than 4%wt fibers. SEM showed better fiber-resin interaction for S groups, and fractures around fibers on N groups. Previous silane application enables the addiction of greater quantity of glass fibers and better interaction with the acrylic resin resulting in higher flexural strength. Without silane, fibers seem to act as initial crack points due to poor interaction.

Modulation of DHPR Inactivation by RyR1 Activity in Mouse Skeletal Muscle Fibers

Malignant hyperthermia is a potentially fatal hypermetabolic state originating from excessive release of calcium stored in the sarcoplasmic reticulum (SR) of skeletal muscle. In most cases, MH susceptibility results from mutations in the type 1 ryanodine receptor (RyR1). In previous work (Andronache et al., PNAS 2009) we reported that heterozygous murine carriers of MH mutation Y524S (human Y522S) exhibit changes in steady state inactivation of the dihydropyridine receptor (DHPR), the sensor of the transverse tubular (TT) membrane potential. Availability curves were left shifted along the voltage axis suggesting that a feedback signal from RyR1 modulates DHPR inactivation. In the present study we investigated the hypothesis that junctional fluctuations of free Ca2+ concentration are involved in the feedback mechanism. We performed two-electrode voltage clamp experiments on enzymatically isolated toe muscle fibers of both WT and mutant mice (Y524S+/−) and measured L-type Ca2+ current and optical signals from fluorescent Ca2+ indicators. To test the hypothesis we applied conditions that would modify junctional Ca2+ levels. Millimolar concentrations of caffeine led to a left shift in the availability curve for L-type current indicating that drug-induced RyR1 hyperactivity can mimic the effect of the mutation. On the other hand, internal dialysis with an artificial solution containing 10 mM of BAPTA to effectively reduce the local Ca2+ transients near open RyR1 channels had little effect on the difference in steady state inactivation between WT and mutant fibers. We conclude that the altered inactivation depends on RyR1 hyperactivity but does not require the continuous presence of local Ca2+ fluctuations within the junctional gap separating TT and SR.

Familial Hypertrophic Cardiomyopathy-Linked Mutation (K104E) in the Myosin Regulatory Light Chain Affects Sarcomeric Structure and Function in Tg-Mice

Structural and functional effects of the familial hypertrophic cardiomyopathy (FHC)-linked mutation (Lysine 104-to-Glutamic Acid, K104E) in the human ventricular regulatory light chain (RLC) were studied in transgenic (Tg) mice. Small angle X-ray diffraction measurements on freshly skinned papillary muscle fibers demonstrated a mutation-induced increase in the interfilament lattice spacing at both short (2.3 µm) and long (2.5 µm) sarcomere lengths (SL) compared with Tg-WT. The intensity ratio (I1,1/I1,0) in WT fibers increased upon stretch (from SL=2.3 to SL=2.5 µm) indicating a shift in the cross-bridge mass distribution toward the thin filaments. In contrary, the K104E fibers demonstrated no change in I1,1/I1,0 on stretch. In line with X-ray data, K104E fibers developed higher passive tension at pCa 8 when consecutively stretched by 10%, 20%, 30% and 40% of their length compared to WT. Furthermore, the K104E mutation significantly decreased the endogenous RLC phosphorylation (∼2-fold) compared to WT myocardium. Histopathological changes included signs of hypertrophy and severe fibrotic lesions in Tg-K104E hearts. The higher mitochondrial content seen in the left ventricles of K104E mice by electron microscopy was supported by significantly increased myosin ATPase activity in Tg-K104E mice indicating higher energy demand and ATP consumption by the mutant hearts. Studies using single molecule detection demonstrated significantly decreased cross-bridge dissociation rates with lower duty cycle (indicative of lower force production) in Tg-K104E cardiac myofibrils compared to WT. Our results in Tg-K104E mice demonstrated cardiomyocyte hypertrophy and fibrosis, and significant alterations in sarcomere structure and function. Morphological abnormalities, increased passive stiffness and a slower cross bridge dissociation rate in Tg-K104E mice mirror the diastolic dysfunction phenotype observed in K104E positive patients. Supported by AHA12PRE12030412 (to WH), and NIH HL-071778 and HL-108343 (to DSC).

Mitochondrial Ca2+-Handling in Fast Skeletal Muscle Fibers from Wild Type and Calsequestrin-Null Mice

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.

From chaos to split‐ups – SHG microscopy reveals a specific remodelling mechanism in ageing dystrophic muscle

Duchenne muscular dystrophy (DMD) is a common inherited muscle disease showing chronic inflammation and progressive muscle weakness. Absent dystrophin renders sarcolemma more Ca(2+) -permeable, disturbs signalling and triggers inflammation. Sustained degeneration/regeneration cycles render muscle cytoarchitecture susceptible to remodelling. Quantitative morphometry was introduced in living cells using second-harmonic generation (SHG) microscopy of myosin. As the time course of cellular remodelling is not known, we used SHG microscopy in mdx muscle fibres over a wide age range for three-dimensional (3D) rendering and detection of verniers and cosine angle sums (CASs). Wild-type (wt) and transgenic mini-dystrophin mice (MinD) were also studied. Vernier densities (VDs) declined in wt and MinD fibres until adulthood, while in mdx fibres, VDs remained significantly elevated during the life span. CAS values were close to unity in adult wt and MinD fibres, in agreement with tight regular myofibril orientation, while always smaller in mdx fibres. Using SHG 3D morphometry, we identified two types of altered ultrastructure: branched fibres and a novel, previously undetected 'chaotic' fibre type, both of which can be classified by distinct CAS and VD combinations. We present a novel model of tissue remodelling in dystrophic progression with age that involves the transition from normal to chaotic to branched fibres. Our model predicts a ~50% contribution of altered cytoarchitecture to progressive force loss with age. We also provide an improved automated image algorithm that is suitable for future ageing studies in human myopathies.

Ciguatoxins activate specific cold pain pathways to elicit burning pain from cooling

Ciguatoxins are sodium channel activator toxins that cause ciguatera, the most common form of ichthyosarcotoxism, which presents with peripheral sensory disturbances, including the pathognomonic symptom of cold allodynia which is characterized by intense stabbing and burning pain in response to mild cooling. We show that intraplantar injection of P-CTX-1 elicits cold allodynia in mice by targeting specific unmyelinated and myelinated primary sensory neurons. These include both tetrodotoxin-resistant, TRPA1-expressing peptidergic C-fibres and tetrodotoxin-sensitive A-fibres. P-CTX-1 does not directly open heterologously expressed TRPA1, but when co-expressed with Na(v) channels, sodium channel activation by P-CTX-1 is sufficient to drive TRPA1-dependent calcium influx that is responsible for the development of cold allodynia, as evidenced by a large reduction of excitatory effect of P-CTX-1 on TRPA1-deficient nociceptive C-fibres and of ciguatoxin-induced cold allodynia in TRPA1-null mutant mice. Functional MRI studies revealed that ciguatoxin-induced cold allodynia enhanced the BOLD (Blood Oxygenation Level Dependent) signal, an effect that was blunted in TRPA1-deficient mice, confirming an important role for TRPA1 in the pathogenesis of cold allodynia.

Impact of pulp rheological behavior on selective separation of Ni minerals from fibrous serpentine ores

Froth flotation based on selective separation of the Ni minerals from ores containing large amounts of fibrous serpentines (chrysotile) has been a long lasting challenge. One underlying reason has largely been considered to be slime-coating. The findings from this investigation, on the other hand, show that the pulp rheological behavior of the flotation pulp is the underlying basic reason. Flotation pulp (25% solids) appeared atypically viscous in the flotation tests carried out with copper ore containing either chrysotile or nylon fibers, where fibers (1–6% by wt fibers) were deliberately added in the grinding stage. With an increase in the pulp fiber (chrysotile and nylon) content, an increase in the pulp yield stress values and a corresponding decrease in the Copper recovery were observed. Notably, pulp was immovable when the copper ore contained more than either 6% of chrysotile or 2% of nylon fibers. Increase in the pulp yield stress values has been proposed to be due to the entanglement of fibers, further resulting in the formation of fiber networks in the pulp. The height, texture, and consistency of the froth phase were observed to be different with increases in the pulp yield stress values. Thus, the above findings evidently suggest that pulp rheological behavior plays a critical role in effective flotation beneficiation of ore containing fibrous minerals. Most importantly, at lower (∼2% by wt in ore) fiber concentrations a subtle change (e.g., increase in pulp yield stress) in rheological behavior may result, however, could possibly contribute to a decrease in the value mineral recovery.

Excitation Coupled Calcium Entry (ECCE) is Enhanced in mdx Myofibers and is Modulated by ROS

Altered Ca2+ homeostasis and elevated ROS have been implicated in the pathogenesis of Duchene Muscular Dystrophy (DMD). In these studies we investigated the contribution of Excitation Coupled Calcium Entry (ECCE) to the altered Ca2+ signaling phenotype and whether this Ca2+ influx pathway is redox sensitive. Adult, post necrotic mdx mice (e.g., > 4 months) exhibited significantly enhanced basal Ca2+ influx at rest compared to WT as determined by Mn2+ quench. Upon depolarization, Ca2+ influx (i.e., ECCE) was significantly enhanced in the mdx compared to WT. Scavenging ROS with n-acetylcysteine (NAC) did not alter the basal Ca2+ influx in the WT or mdx muscle fibers. NAC, however, significantly reduced ECCE in mdx muscle by approximately 66% with no effect on WT. The non-specific TRP channel inhibitor 4-methy-4¯-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]-1,2,3-thiadiazole-5-carboxanilide (BTP-2) reduced basal Ca2+ influx and ECCE in mdx muscle fibers by 51% and 72% respectively. In WT, BTP2 reduced ECCE by 41% and had no effect on basal Ca2+ permeability. We conclude that basal Ca2+ influx and ECCE are likely TRP dependent and only ECCE in the mdx can be modulated by ROS scavenging. Funding: LPM - 5F31NR011245-03, CWW- RC2 NR011968, GGR-K01AR051519

Different Capacity for Store-Operated Ca2+ Entry and Ca2+ Extrusion Across the Plasma Membrane of Wild-Type and Dystrophic mdx Mouse Muscle

Store-operated Ca2+ entry (SOCE) is a ubiquitously expressed signalling system that is highly specialized in skeletal muscle. “Deregulated” SOCE had been proposed as a pathway for Ca2+ entry into dystrophic muscle that leads to fibre degradation. We recently showed that this mechanism remains tightly regulated in mdx mouse muscle but the integral SOCE proteins, STIM1 and Orai1, are upregulated 3-fold (Edwards et al., 2010). We now report that the newly identified isoform STIM1L (Darbellay et al., 2011) is upregulated 1.8-fold in mdx muscle. We found SOCE recorded in skinned fibres was 2-fold greater in mdx compared with WT for the same SR Ca2+ release amplitude. However cytoplasmic fluo-4 transients in depleted intact fibres showed SOCE in the absence of a sarcoplasmic reticulum (SR) Ca2+ pump blocker to be of reduced influx rate in mdx compared to WT fibres. A similar level of SR Ca2+ reloading was detemined in both muscle types following SOCE deactivation. Fura-2 imaging in intact fibres in the presence of 50 μM cyclopiazonic acid (CPA) and no external Ca2+ showed that more Ca2+ remained in the cytoplasm of mdx compared to WT fibres following SR depletion suggesting that Ca2+ extrusion by the plasma membrane Ca2+-ATPase (PMCA) is restricted in mdx. This helps explain reduced SOCE in intact mdx fibres, as the washout of CPA following SR depletion resulted in the greater amount of trapped cytoplasmic Ca2+ re-entering SR to cause a greater degree of SOCE deactivation before externally applied Ca2+ could enter the fibre. These results suggest that store-dependent Ca2+ influx is greater and PMCA is restricted in its capacity to extrude Ca2+ in mdx compared to WT fibres.

Extracellular Adaptations to Altered Force Transmission in Muscle Cells

Interaction between active force generating components and passive extracellular matrix (ECM) force transmitting components provides skeletal muscle with its characteristic biomechanical properties, enabling it to produce work and resist extension. However, the mechanisms behind this “communication” and the cellular structures that enable it are poorly understood. Desmin is an intermediate filament protein integral to the muscle fiber cytoskeleton. It is thought to be involved in stabilizing the contractile apparatus and transmitting force between the cytoskeleton and the ECM. In this study, we investigated the effects of desmin deletion (des-/-) on the composition and mechanical properties of the cellular and extracellular components of muscle.We found that, while des-/- and wt muscles have identical fiber and bundle (fibers+ECM) material properties at birth, these properties diverge becoming disparate with age. Specifically, des-/- fibers become more compliant compared to wt fibers (tangent modulus: 42.8±6 kPa/um and 66.8±7 kPa/um respectively), while des-/- bundles become progressively stiffer compared to wt (tangent modulus: 193.3±19 kPa/um and 129±13 kPa/um respectively). This result suggests that des-/- muscles are chronically altering their ECM. Evidence supporting this hypothesis was provided by significantly increased collagen protein content (des-/-: 8.0±1 ug/mg, wt: 3.4±1 ug/mg), ECM area fraction (des-/-: 15.1±1%, wt: 10.5±1%) and fibrosis-related gene expression patterns in des-/- muscle compared with wt. Additionally, significantly increased numbers of centrally nucleated fibers (des-/-: 5.6±0.5%, wt: 0.7±0.2%) and inflammatory gene expression patterns in des-/- muscle suggest that an increased susceptibility to injury in des-/- fibers may underlie the measured fibrotic response. Thus, in this study, we identify a possible causal relationship between increased cellular compliance, due to reduction in force transmission and stabilization by desmin, and the proliferative response of the extracellular matrix cellular constituents.