Flexor Digitorum Brevis Research Articles

The fourth slip of the flexor digitorum brevis muscle of the human foot. A systematic review and meta-analysis.

The flexor digitorum brevis muscle, and in particular its fourth slip, has a significant clinical and surgical importance in medical practice. However, as for the majority of tendons destined to the little toe, the fourth slip is undergoing a phylogenetic degeneration. The aim of this meta-analysis is to conduct an evidence synthesis on the prevalence of FDB-5 and its variants in humans. Twenty-two studies met pre-defined inclusion criteria with a total of 2789 feet and 416 cadavers. Meta-analytical results of fourth slip agenesis were as follows; a) a true prevalence rate of 31.3%, b) a crude prevalence rate of 47%, c) a bilateral prevalence rate of 38.2%, d) a true prevalence rate of 77.3% in Indian populations, e) a true prevalence rate of ≈ 20% in Japanese, Chinese, American, European and Turkish populations, f) an Odds Ratio of 1.5 significantly in favor for female gender, g) non-significance for hand side, h) a true prevalence rate of a thin fourth slip of 47.7%, and i) a true prevalence rate of the variations of its origin in 12.3%. The knowledge of the frequency of flexor digitorum brevis fourth slip agenesis and variations in relation to the demographic characteristics of patients would be of importance for tendon repair, tendon transfer or soft tissue reconstruction in foot surgery.

Active regulation of longitudinal arch compression and recoil during walking and running.

The longitudinal arch (LA) of the human foot compresses and recoils in response to being cyclically loaded. This has typically been considered a passive process, however, it has recently been shown that the plantar intrinsic foot muscles have the capacity to actively assist in controlling LA motion. Here we tested the hypothesis that intrinsic foot muscles, abductor hallucis (AH), flexor digitorum brevis (FDB) and quadratus plantae (QP), actively lengthen and shorten during the stance phase of gait in response to loading of the foot. Nine participants walked at 1.25 m s⁻¹ and ran at 2.78 and 3.89 m s⁻¹ on a force-instrumented treadmill while foot and ankle kinematics were recorded according to a multisegment foot model. Muscle-tendon unit (MTU) lengths, determined from the foot kinematics, and intramuscular electromyography (EMG) signals were recorded from AH, FDB and QP. Peak EMG amplitude was determined during the stance phase for each participant at each gait velocity. All muscles underwent a process of slow active lengthening during LA compression, followed by a rapid shortening as the arch recoiled during the propulsive phase. Changes in MTU length and peak EMG increased significantly with increasing gait velocity for all muscles. This is the first in vivo evidence that the plantar intrinsic foot muscles function in parallel to the plantar aponeurosis, actively regulating the stiffness of the foot in response to the magnitude of forces encountered during locomotion. These muscles may therefore contribute to power absorption and generation at the foot, limit strain on the plantar aponeurosis and facilitate efficient foot ground force transmission.

Sarcolemmal Biomechanics and Excitability in Malformed Muscle Fibers of Dystrophic Mice

Background: Duchenne muscular dystrophy (DMD), the most common and severe dystrophy, is caused by the absence of dystrophin. Muscle weakness and fragility (i.e. increased susceptibility to damage) are presumably due to structural weakness of the myofiber cytoskeleton, but recent studies suggest that malformed/split myofibers in dystrophic muscle may also play a role. We have previously studied the biomechanical properties of the sarcolemma in single myofibers isolated mechanically from extensor digitorum longus (EDL) muscles in wild-type (WT) and dystrophic (mdx, mouse model for DMD) mice. PURPOSE: We use similar biomechanical methods on enzymatically-dissociated myofibers (both normal and malformed) from the flexor digitorum brevis muscle (FDB) of WT and mdx mice.Methods: FDB muscles were enzymatically-dissociated and plated on specialized coverslips. Suction pressures (P) applied through a pipette to the membrane generated a bleb, which increased in height with increasing P. Larger increases in P ruptured the connections between the sarcolemma and myofibrils (speration P, or SP) and eventually caused the sarcolemma to burst. We also examined excitability using high-speed confocal microscopy and the voltage-sensitive indicator di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate to assess the action potential (AP).Results: The mechanical results from dissociated FDB myofibers match findings from dissected EDL myofibers, but SP was up to 14-fold higher in the FDB than EDL. SP was 27% lower in mdx myofibers and 50% less in branches of split mdx fibers compared to the trunk. AP amplitude was not altered in between groups, but this work is ongoing.Conclusions: Data indicate a reduction in muscle stiffness, increased sarcolemmal deformability and instability in mdx muscle. This approach corroborates the labor-intensive data obtained from single fiber dissection and allows a facile high throughput model. Findings suggest mechanical differences due to altered morphology, despite comparable excitability.

Calcium Channel Dysfunction in a Mutant Mouse Model of Malignant Hyperthermia(CaV1.1 R174W)

Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic disorder of skeletal muscle that is triggered by exposure to volatile anesthetics. MH has been studied extensively in mice and pigs carrying causative mutations in the type 1 ryanodine receptor (RyR1). However, no in vivo information exists regarding how mutations in the skeletal muscle L-type Ca2+ channel (CaV1.1) precipitate MH crises. For this reason, we generated a mouse line carrying the R174W mutation. Homozygous R174W mice ambulated efficiently, reproduced and had normal lifespans. When exposed to isoflurane, homozygous R174W mice entered a hypermetabolic state ending ultimately in death. On the ultrastructural level, R174W muscle displayed limited and variable changes: some variability of the SR calsequestrin content, displacement of mitochondria in some soleus fibers of aged mice and occasional accumulation of SR stacks. On the cellular level, homozygous R174W muscle had elevated resting myoplasmic Ca2+ levels that were greatly increased upon exposure to isoflurane. Flexor digitorum brevis (FDB) fibers dissociated from homozygous R174W mice lacked L-type Ca2+ current even though intramembrane charge movements of were of similar magnitude and voltage-dependence to those recorded from wild-type fibers. Ca2+ released from the SR in response to depolarization was substantially reduced in homozygous R174W fibers suggesting a depleted SR Ca2+ store. Lipid bilayer recordings showed that the Po of RyR1s isolated from homozygous R174W mice was significantly increased at all cis Ca2+ concentrations (Feng et al., this meeting). Taken together, our results support a mechanism for MH susceptibility in which CaV1.1 R174W promotes SR Ca2+ leak without affecting EC coupling per se. This work was supported by grants from the NIH (AR055104 to KGB, AR052534 to PDA, KGB, PMH, CFA and INP) and MDA (MDA277475 to KGB). DB received a stipend from 2T32AG000279-11.

Spatially Localized Disruptions of Voltage Activated Calcium Release in Mtm1-Deficient Muscle Fibers

Mutations in the gene encoding the phosphoinositide phosphatase myotubularin (Mtm1) are responsible for myotubular myopathy. We previously showed that muscle fibers from Mtm1-deficient mouse suffer from defective excitation-contraction coupling. Here we measured voltage-clamp activated Ca2+ signals in fibers from the flexor digitorum brevis muscles of 4 week-old wild type (WT) and Mtm1-ko mice under line-scan confocal microscopy using the dye rhod-2. Ca2+ release in the diseased fibers was deficient over the full range of activation: fitting the voltage dependence of the peak rate of rise of the line-averaged rhod-2 F/F0 signals with a Boltzmann function gave mean values for maximum rate, midpoint voltage and slope of 0.29 ± 0.02 and 0.14 ± 0.02 F/F0.ms−1, −8.8 ± 1.5 and 0.95 ± 2.3 mV, 7.0 ± 0.4 and 9.2 ± 0.7 mV in WT (n=22) and Mtm1-ko fibers (n=15), respectively. Furthermore, the mean time to peak rate was significantly delayed by 5-10 ms in Mtm1-ko as compared to WT fibers. These global alterations were associated with severe spatial inhomogeneity of Ca2+ release in the diseased fibers with rhod-2 transients yielding localized disruptions along the scanned line including reduced peak amplitude but also delayed or slower rate of onset, suggestive of specific alteration of the early peak component of the rate of Ca2+ release. In fibers treated with wortmannin and LY294002, the properties of the line-averaged rhod-2 F/F0 signals were unchanged in WT fibers but the mean maximum rate of rise of the rhod-2 signal was enhanced by 60% in Mtm1-deficient fibers. Results show that Mtm1-deficiency provokes EC coupling failure through accumulation of spatially localized disruptions of Ca2+ release. They also suggest that pharmacological inhibition of PtdIns-3-kinase activity has the potency to alleviate defective EC coupling in the diseased fibers.

Characterization of Dual Mutant RyR1D-S100A1KO Mice with Disrupted CaM and S100A1 Binding to CaMBD2 and Lacking S100A1 Expression

S100A1 and calmodulin (CaM) modulate skeletal muscle Ca2+ signaling. Recently, isothermal titration calorimetry demonstrated that CaM binds to three regions within the ryanodine receptor type-1 (RyR1) sequence: CAMBD1, residues 1975-1999; CaMBD2, residues 3614-3640; CAMBD3, residues 4295-4325 (Lau K, et al., 2014). We previously studied the binding to and modulation of RyR1 by CaM and S100A1 via CaMBD2, showing that skeletal muscle fibers from S100A1 knock out (S100A1KO) mice or RyR1-L3625D (RyR1D) mice (having disrupted CaM and S100A1 binding to CaMBD2), exhibit impaired RyR1 regulation. Here we propose and test the hypothesis that S100A1 also modulates fiber activation via sites other than CaMBD2. Double mutant mice were generated by crossbreeding homozygous RyR1D and S100A1KO mice to obtain homozygous double mutant RyR1D-S100A1KO mice, which were viable and appeared normal. Homozygous RyR1D mice were used as controls to eliminate any effect of binding at CaMBD2. In vivo stimulation of tibialis anterior muscles in anesthetized mice showed that maximal specific force was reduced in RyR1D-S100A1KO compared with RyR1D muscles (RyR1D Po= 5.15 ± .09 g/mg, RyR1D-S100A1KO Po= 4.05 ± .09 g/mg; p < 0.05). Using high-speed confocal microscopy of Ca2+ indicator rhod-2, we show that RyR1D-S100A1KO flexor digitorum brevis muscle fibers exhibit increased global myoplasmic Ca2+ transients following field stimulation. We also found no differences in myoplasmic resting Ca2+ between RyR1D and RyR1D-S100A1KO. Our results indicate that RyR1D-S100A1KO muscles have increased SR Ca2+ release, however exhibit reduced force generation. These results suggest that S100A1, similar to CaM, binds to different regions within the RyR1, but also indicates that S100A1 could bind to other target proteins (i.e., contractile apparatus). Where and how exactly S100A1 exerts these additional effects is currently unknown. Support: NIH R37-AR055099 and R01-AR059179.

Mir181A Targets the 3'UTR of MG29, a Muscle-Specific Synaptophysin Family Gene, for Down-Regulation of MG29 Expression in Dystrophic Skeletal Muscle

MG29 is a muscle-specific member of the synaptophysin family protein that participates in controlling the maturation and development of transverse tubule (TT) structure and the maintenance of intracellular Ca2+ signaling in skeletal muscle. Genetic ablation of MG29 leads to defective formation of the TT network in skeletal muscle, which resembles the abnormal TT structure observed in dystrophic muscles. We found that MG29 protein is drastically reduced in mouse models with muscular dystrophy (e.g. mdx and dysferlin-/-), and such reduced MG29 expression and disrupted TT structure were also observed in human patient biopsies with muscular dystrophy. The mg29 gene contains a unique 3’ untranslated region (UTR) with potential binding sites for microRNA (miRNA) or RNA-stabilizing factors. Mutagenesis studies revealed that miR-181a could target a region in the 3′UTR that is highly conserved between mouse and human mg29 genes to exhibit posttranscriptional control of MG29 expression in skeletal muscle. Overexpression of anti-sense miR-181a by microinjection into mouse flexor digitorum brevis fibers significantly increased the expression of endogenous MG29. Studies from other investigators have shown that miR-181a was elevated in human patients with muscular dystrophy. These data suggest an intriguing possibility that elevated miR-181a may be a contributing factor for the reduced MG29 expression under dystrophic conditions. Further understanding of the post-transcriptional regulation by microRNA of MG29 expression can provide more insights into the therapeutic targets of muscular dystrophy.

Malignant Hyperthermia Susceptibility Mutation Cav1.1 R174W Dramatically Alters RyR1 Single Channel Function

Nearly 200 variants in the gene encoding the skeletal muscle RyR1 Ca2+ channel are associated with MHS but only 30 were demonstrated to perturb channel function. We previously showed that mouse MHS mutations RyR1_R163C or RyR1_T4826I have abnormally high open probabilities and elevated [3H]ryanodine binding when compared to wild type. Recently, a small number of MHS families were shown to express mutations, not in the RyR1, but rather in the gene encoding the skeletal muscle L-type Ca2+ channel (CaV1.1). We therefore generated a knock-in mouse line expressing CaV1.1_R174W that confers MHS in human kindred. Homozygous (HOM) mice survive, but trigger with fulminant MH when exposed to halogenated volatile anaesthetics, have chronically elevated resting myoplasmic Ca2+, and lack L-type Ca2+ current in adult Flexor digitorum brevis fibers (Bannister et al, this meeting). It is unknown however if MHS mutations residing within CaV1.1 are capable of modifying RyR1 structure/function thereby contributing to disrupted intracellular Ca2+ homeostasis. To address this question, SR membranes were prepared from the skeletal muscles of age-matched WT and HOM Cav1.1_R174W for biochemical and electrophysiological analysis. Unexpectedly, we found significantly abnormal properties of HOM as compared to WT in: (1) enhanced [3H]Ry binding; (2) elevated levels of RyR1/FKBP12 expression, and (3) exceptionally higher Po with channels in BLM at all [Ca2+]cis. Even at [Ca2+]cis=120nM, an inhibitory condition to RyR1 in normal WT muscle fibers, RyR1 channels from CaV1.1_R174W muscle maintained a conformation accessible for ryanodine binding. These results demonstrate that a MHS mutation residing outside of RyR1 is capable of persistently modifying RyR1 channel function. These findings have significant implications about the molecular mechanisms that lead to MHS and fulminant MH. (Grant supports: AR052534 to PDA, KGB, PMH, CFA, INP; AR055104 and MDA277475 to KGB).

The cholesterol-lowering agent methyl-β-cyclodextrin promotes glucose uptake via GLUT4 in adult muscle fibers and reduces insulin resistance in obese mice.

Insulin stimulates glucose uptake in adult skeletal muscle by promoting the translocation of GLUT4 glucose transporters to the transverse tubule (T-tubule) membranes, which have particularly high cholesterol levels. We investigated whether T-tubule cholesterol content affects insulin-induced glucose transport. Feeding mice a high-fat diet (HFD) for 8 wk increased by 30% the T-tubule cholesterol content of triad-enriched vesicular fractions from muscle tissue compared with triads from control mice. Additionally, isolated muscle fibers (flexor digitorum brevis) from HFD-fed mice showed a 40% decrease in insulin-stimulated glucose uptake rates compared with fibers from control mice. In HFD-fed mice, four subcutaneous injections of MβCD, an agent reported to extract membrane cholesterol, improved their defective glucose tolerance test and normalized their high fasting glucose levels. The preincubation of isolated muscle fibers with relatively low concentrations of MβCD increased both basal and insulin-induced glucose uptake in fibers from controls or HFD-fed mice and decreased Akt phosphorylation without altering AMPK-mediated signaling. In fibers from HFD-fed mice, MβCD improved insulin sensitivity even after Akt or CaMK II inhibition and increased membrane GLUT4 content. Indinavir, a GLUT4 antagonist, prevented the stimulatory effects of MβCD on glucose uptake. Addition of MβCD elicited ryanodine receptor-mediated calcium signals in isolated fibers, which were essential for glucose uptake. Our findings suggest that T-tubule cholesterol content exerts a critical regulatory role on insulin-stimulated GLUT4 translocation and glucose transport and that partial cholesterol removal from muscle fibers may represent a useful strategy to counteract insulin resistance.

Role of Mitofusin-2 in mitochondrial localization and calcium uptake in skeletal muscle

As muscle contraction requires ATP and Ca2+, skeletal muscle function is highly dependent on communication between two major intracellular organelles: mitochondria and sarcoplasmic reticulum (SR). In adult skeletal muscle, mitochondria located within the I-band of the sarcomere are connected to the SR by small ∼10nm electron dense tethers that bridge the outer mitochondrial membrane to the region of SR that is ∼130nm from the site of Ca2+ release. However, the molecular composition of tethers and their precise impact on mitochondrial Ca2+ uptake in skeletal muscle is unclear. Mitofusin-2 (Mfn2) is a transmembrane GTPase present in both mitochondria and ER/SR membranes that forms trans-dimers and participates in mitochondrial fusion. Here we evaluated the role Mfn2 plays in mitochondrial morphology, localization, and functional SR-mitochondrial Ca2+ crosstalk in adult skeletal muscle. Compared to a non-targeting (CTRL) siRNA, in vivo electroporation of 400nM Mfn2 siRNA (Mfn2 KD) into mouse footpads resulted in a marked acute reduction (67±3%) in Mfn2 protein levels in flexor digitorum brevis (FDB) muscles that occurred without a change in other key Ca2+ regulatory proteins. Electron microscopy analyses revealed that Mfn2 knockdown resulted in a change in mitochondria morphology and mis-localization of some mitochondria from the I-band to the A-band region of the sarcomere. To assess the role of Mfn2 in SR-mitochondrial crosstalk, we measured mitochondrial Ca2+ uptake and myoplasmic Ca2+ transients with rhod-2 and mag-fluo-4, respectively, during repetitive high frequency tetanic stimulation (5×100Hz tetani, 500ms/tetani, 0.2 duty cycle) in CTRL and Mfn2 KD fibers. Mitochondrial Ca2+ uptake during repetitive tetanic stimulation was significantly reduced (40%) in Mfn2 KD FDB fibers, which was accompanied by a parallel elevation in the global electrically evoked myoplasmic Ca2+ transient. Mfn2 KD also resulted in a reduction of the mitochondrial membrane potential, which contributed to the observed decrease in activity-dependent mitochondrial Ca2+ uptake. Consistent with this idea, a similar decrease in mitochondrial Ca2+ uptake was also observed in wild type fibers following a comparable reduction in mitochondrial membrane potential induced by acute exposure to a low concentration (50nM) of carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP). In addition, both global and mitochondrial Ca2+ transients during repetitive tetanic stimulation were similarly reduced by both slow (EGTA) and fast (BAPTA) Ca2+ chelating agents. Together, these results indicate that Mfn2 promotes proper mitochondrial morphology, localization, and membrane potential required for optimal activity-dependent mitochondrial Ca2+ uptake and buffering of the global myoplasmic Ca2+ transient in adult skeletal muscle.

Accurate segmental motor innervation of human lower-extremity skeletal muscles.

There is limited knowledge about accurate segmental motor innervation of the human lower extremity skeletal muscles. The aim of the present study was to explore the truth of segmental motor innervation of the lower extremity. Included in this study were 20 patients with unilateral fracture of the sacrum and sacral nerve injury, who underwent internal fixation and decompression of the sacral nerve in our hospital between June 2009 and January 2014. L4-S4 nerve roots of the uninjured side were stimulated during operation. Motor innervation was determined by stimulating the spinal nerves with supramaximal intensity. We found the gluteus medius and the gluteus maximus were both mainly innervated by L5 and S1. In addition, the nerve fibres that innervated the extensor digitorum brevis, the abductor hallucis and the flexor digitorum brevis were mainly from S2 to S3. Our study provides the electrophysiological mapping of the segmental anatomy of the human lower extremity muscles, which should be clinically useful in helping the diagnosis and treatment of nerve injury and neuropathies.

Monovalent Cationic Channel Activity in the Inner Membrane of Nuclei from Skeletal Muscle Fibers

Nuclear ion channels remain among the least studied and biophysically characterized channels. Although considerable progress has been made in characterizing calcium release channels in the nuclear membrane, very little is known regarding the properties of nuclear monovalent cationic channels. Here, we describe a method to isolate nuclei from adult skeletal muscle fibers that are suitable for electrophysiological experiments. Using this approach, we show for the first time, to our knowledge, that a nuclear monovalent cationic channel (NMCC) is prominently expressed in the inner membrane of nuclei isolated from flexor digitorum brevis skeletal muscle fibers of adult mice. In isotonic 140 mM KCl, the skeletal muscle NMCC exhibits a unitary conductance of ∼160 pS and high, voltage-independent open probability. Based on single-channel reversal potential measurements, NMCCs are slightly more permeable to potassium ions over sodium (PK/PNa = 2.68 ± 0.21) and cesium (PK/PCs = 1.39 ± 0.03) ions. In addition, NMCCs do not permeate divalent cations, are inhibited by calcium ions, and demonstrate weak rectification in asymmetric Ca2+-containing solutions. Together, these studies characterize a voltage-independent NMCC in skeletal muscle, the properties of which are ideally suited to serve as a countercurrent mechanism during calcium release from the nuclear envelope.

The role of proteases in excitation-contraction coupling failure in muscular dystrophy.

Duchenne muscular dystrophy (DMD) is one of the most frequent types of muscular dystrophy. Alterations in intracellular calcium (Ca(2+)) handling are thought to contribute to the disease severity in DMD, possibly due to the activation of Ca(2+)-activated proteases. The purpose of this study was twofold: 1) to determine whether prolonged excitation-contraction (E-C) coupling disruption following repeated contractions is greater in animals lacking both dystrophin and utrophin (mdx/Utr(-/-)) compared with mice lacking only dystrophin (mdx); and 2) to assess whether protease inhibition can prevent E-C coupling failure following repeated tetani in these dystrophic mouse models. Excitation-contraction coupling was assessed using Fura-2 ratio, as an index of intracellular free Ca(2+) concentration, in response to electrical stimulation of single muscle fibers from the flexor digitorum brevis muscle. Resting Fura-2 ratio was higher in dystrophic compared with control (Con) fibers, but peak Fura-2 ratios during stimulation were similar in dystrophic and Con fibers. One hour after a series of repeated tetani, peak Fura-2 ratios were reduced by 30 ± 5.6%, 23 ± 2%, and 36 ± 3.1% in mdx, mdx/Utr(+/-), and mdx/Utr(-/-), respectively, with the greatest reduction in mdx/Utr(-/-) fibers (P < 0.05). Protease inhibition attenuated this decrease in peak Fura-2 ratio. These data indicate that E-C coupling impairment after repeated contractions is greatest in fibers lacking both dystrophin and utrophin and that prevention of protease activation can mitigate the prolonged E-C coupling impairment. These data further suggest that acute protease inhibition may be useful in reducing muscle weakness in DMD.

Force enhancement after stretch in mammalian muscle fiber: no evidence of cross-bridge involvement.

Stretching of activated skeletal muscles induces a force increase above the isometric level persisting after stretch, known as residual force enhancement (RFE). RFE has been extensively studied; nevertheless, its mechanism remains debated. Unlike previous RFE studies, here the excess of force after stretch, termed static tension (ST), was investigated with fast stretches (amplitude: 3-4% sarcomere length; duration: 0.6 ms) applied at low tension during the tetanus rise in fiber bundles from flexor digitorum brevis (FDB) mouse muscle at 30°C. ST was measured at sarcomere length between 2.6 and 4.4 μm in normal and N-benzyl-p-toluene sulphonamide (BTS)-added (10 μM) Tyrode solution. The results showed that ST has the same characteristics and it is equivalent to RFE. ST increased with sarcomere length, reached a peak at 3.5 μm, and decreased to zero at ∼4.5 μm. At 4 μm, where active force was zero, ST was still 50% of maximum. BTS reduced force by ∼75% but had almost no effect on ST. Following stimulation, ST developed earlier than force, with a time course similar to internal Ca(2+) concentration: it was present 1 ms after the stimulus, at zero active force, and peaked at ∼3-ms delay. At 2.7 μm, activation increased the passive sarcomere stiffness by a factor of ∼7 compared with the relaxed state All our data indicate that ST, or RFE, is independent of the cross-bridge presence and it is due to the Ca(2+)-induced stiffening of a sarcomeric structure identifiable with titin.

G.O.9: AAV gene transfer utilizing homologous overlap vectors mediates functional recovery of dysferlin deficiency

There is no cure for dysferlinopathies, a group of related disorders caused by absent or mutant dysferlin (DYSF). Lack of dysferlin leads to progressive dystrophy with chronic muscle fiber loss, inflammation, fat replacement and fibrosis leading to deteriorating muscle weakness. Dysferlin has multiple functions including T-tubule formation and membrane repair. Pre-clinical studies show that only delivery of full-length DYSF corrects the histopathological defects in DYSF−/− mice. As DYSF is too large to be accommodated with canonical packaging, we developed a unique dual vector system using AAV to deliver and express DYSF in muscle. This two vector system (AAV.DYSF.DV) packaged in the rh.74 serotype is defined by a 1 kb region of homology between the two vectors. We compared the efficiency and safety of DYSF.DV delivery in dysferlin deficient mice and in non-human primates (NHPs) in preparation for clinical trials. Dysf−/− mice were treated by intramuscular injection, isolated limb perfusion, and systemic injection with AAV.DYSF.DV. IM injections were performed in the flexor digitorum brevis (FDB) muscle to assess expression and restoration of membrane repair capacity using ascending vector doses (6e9–6e10vg). The FDB was extracted after 10 wks and an ex vivo laser injury membrane repair assay was performed. DYSF.DV restored repair capacity in a dose-dependent manner comparable to WT findings. DYSF.DV was also efficacious when delivered through arterial circulation to hind limbs or systemically via the tail vein. A systemic dose of 6e12vg resulted in widespread gene expression exceeding 50% of muscle fibers; restoring functional deficits in the diaphragm. In NHPs, DYSF.DV delivery was analyzed for gene expression and toxicity using FLAG tag. There was no evidence of toxicity in DYSF-null mice or NHPs showing overexpression of dysferlin. Findings showed clear evidence that functional dysferlin was translated without toxicity laying the foundation for clinical trial.

G.P.289: Novel ANO5 KO mouse provides new insight into LGMD2L pathogenesis

Limb girdle muscular dystrophy type 2L (LGMD2L) is caused by mutations in the anoctamin 5 (ANO5) gene. ANO5 encodes a predicted 107kDa protein containing 8 transmembrane domains and is part of a family that encodes at least 2 calcium-activated chloride channels; however the exact function of ANO5 has yet to be determined. The clinical phenotype overlaps with that of LGMD2B patients having mutations in dysferlin, a gene required for membrane repair. We have developed an ANO5 deficient mouse model (ANO5 KO) for LGMD2L for which no model currently exists. This model is necessary to study disease pathophysiology and test potential therapies. This deficient mouse model has been fundamental to characterize disease pathogenesis, discovering several proteins that may be associated with ANO5, and related functional incapacities. Based on the phenotypic overlap with dysferlin deficiency, we tested whether our ANO5 KO mouse model had a deficit in muscle membrane repair. Following laser induced injury, the flexor digitorum brevis muscles from ANO5 KO mice demonstrated reduced capacity to repair compared to WT strain controls. In addition, we also showed a loss of force compared to wild type mice after mechanical repetitive eccentric contraction in the extensor digitorum longus muscle.. Furthermore, we identified histopathological changes along with mislocalization or reduction in several muscle proteins which are linked and/or secondary to AN05. The ANO5 deficient mouse is novel and represents an important animal model for study of LGMD2L. It is the ideal model to develop proof of principle for gene replacement therapy or pharmacologic approaches and permits studies defining ANO5 function, a critical parameter for therapeutic intervention. The functional deficits we identified provide critical outcome measures to test efficacy of potential therapies and demonstrate feasibility of translation.

Quantifying the Contributions of a Flexor Digitorum Brevis Muscle on Postural Stability.

Surprisingly little attention has been devoted to the role played by the intrinsic muscles of the human foot. The aim of this study was to quantify the capabilities of the flexor digitorum brevis (FDB) muscle to contribute to upright postural control. The approaches consisted of analysis of the effects of FDB contraction elicited by external electrical stimulation and quantification of the magnitude of FDB torque generation. The results showed the FDB can produce significant changes in static posture by itself as shown by changes in the center of pressure. Moreover, the FDB contribution to counterbalance the gravity's toppling force was estimated at around 14.5% of the total required active torque at the ankle to keep the subject from falling. A posteriori functional analysis during horizontal perturbations showed high and self-sustained activity of FDB. These results demonstrated that the FDB has a significant capability of contributing to postural control.

Medial plantar flap to repair defects of palm volar skin.

The methods of repairing defects in fingers, volar skin of the palm and soft tissue were investigated. From 2010 to the present, we examined 12 cases in which medial plantar skin flaps were used to repair defects in the fingers and palm. According to skin and soft tissue defects in the fingers and palm, a flap was designed using the medial plantar artery as the vessel pedicle. The flap was dissected and isolated between the abductor hallucis and flexor digitorum brevis, and transplanted to the hand. We then observed the skin colour, skin texture and tactile sensitivity of the hand, as well as the shape and function of the foot. Follow-up for 3-28 months showed that the flaps survived in all 12 cases, with soft skin, healthy appearance, colour consistent with the palm skin and no pigmentation. The two-point discrimination was 5-7 mm. The donor foot functioned well, and the scar at the donor site was slight and had an aesthetic appearance. The free medial plantar flap is an ideal flap for repairing soft tissue defects in the palm.

Skeletal muscle contractions induce acute changes in cytosolic superoxide, but slower responses in mitochondrial superoxide and cellular hydrogen peroxide.

Skeletal muscle generation of reactive oxygen species (ROS) is increased following contractile activity and these species interact with multiple signaling pathways to mediate adaptations to contractions. The sources and time course of the increase in ROS during contractions remain undefined. Confocal microscopy with specific fluorescent probes was used to compare the activities of superoxide in mitochondria and cytosol and the hydrogen peroxide content of the cytosol in isolated single mature skeletal muscle (flexor digitorum brevis) fibers prior to, during, and after electrically stimulated contractions. Superoxide in mitochondria and cytoplasm were assessed using MitoSox red and dihydroethidium (DHE) respectively. The product of superoxide with DHE, 2-hydroxyethidium (2-HE) was acutely increased in the fiber cytosol by contractions, whereas hydroxy-MitoSox showed a slow cumulative increase. Inhibition of nitric oxide synthases increased the contraction-induced formation of hydroxy-MitoSox only with no effect on 2-HE formation. These data indicate that the acute increases in cytosolic superoxide induced by contractions are not derived from mitochondria. Data also indicate that, in muscle mitochondria, nitric oxide (NO) reduces the availability of superoxide, but no effect of NO on cytosolic superoxide availability was detected. To determine the relationship of changes in superoxide to hydrogen peroxide, an alternative specific approach was used where fibers were transduced using an adeno-associated viral vector to express the hydrogen peroxide probe, HyPer within the cytoplasmic compartment. HyPer fluorescence was significantly increased in fibers following contractions, but surprisingly followed a relatively slow time course that did not appear directly related to cytosolic superoxide. These data demonstrate for the first time temporal and site specific differences in specific ROS that occur in skeletal muscle fibers during and after contractile activity.

The effect of minimal shoes on arch structure and intrinsic foot muscle strength

BackgroundThis prospective study explored the effects of endurance running (ER) in minimal versus standard running shoes on the foot's superficial layer intrinsic muscles and the function of the longitudinal arch. Our hypothesis was that running in minimal shoes would cause hypertrophy in these muscles and lead to higher, stronger, stiffer arches. MethodsThe hypothesis was tested using a sample of 33 healthy runners randomized into two groups, a control group shod in traditional running footwear and an experimental group shod in minimal support footwear, whose feet were scanned in an MRI before and after a 12-week training regime. Running kinematics as well as arch stiffness and height were also assessed before and after the treatment period. ResultsAnalysis of anatomical cross-sectional areas and muscle volumes indicate that the flexor digitorum brevis muscle became larger in both groups by 11% and 21%, respectively, but only the minimally shod runners had significant areal and volumetric increases of the abductor digiti minimi of 18% and 22%, respectively, and significantly increased longitudinal arch stiffness (60%). ConclusionThese results suggest that endurance running in minimal support footwear with 4 mm offset or less makes greater use of the spring-like function of the longitudinal arch, thus leading to greater demands on the intrinsic muscles that support the arch, thereby strengthening the foot.