Flexor Digitorum Brevis Fibers Research Articles

Unraveling the evolutionary marathon: improved mitochondrial bioenergetics in CMP-Neu5Ac hydroxylase gene deleted (Cmah−/−) mouse skeletal myofibers

Objective: Humans are unique among hominids in that they are endurance athletes. Inactivation of the CMP-Neu5Ac hydroxylase (CMAH) gene, which encodes a hydroxylase converting sialic acid Neu5Ac to Neu5Gc, is thought to contribute to this exceptional capacity for endurance running. Recently, our group demonstrated improved fatigue resistance and preserved intracellular O2, estimated by NAD(P)H levels in contracting mouse Cmah−/− flexor digitorum brevis (FDB) single fibers under low oxygen tension. Hypothesis: Effcient regulation of mitochondrial oxygen consumption contributes to improved skeletal myofiber fatigue resistance in Cmah−/− mice. Methods: The mitochondrial bioenergetic potential of permeabilized FDB and soleus fiber bundles from wild type (WT) and Cmah−/− mice was evaluated using a physiological creatine kinase clamp method and high-resolution respirometer. Experiments were conducted with pyruvate and malate with or without branched chain amino acids (BCAAs) as the substrates. Respiratory chain conductance and maximal respiration were measured. Results: FDB Cmah−/− fibers from male but not female mice revealed an increase conductance above WT fibers independent of substrate (male: genotype, p=0.006, substrate, ns; female: genotype, ns, substrate, ns). Maximal respiration was also increased only in male FDBs fibers and was even greater when BCAAs were included in the respiration media. (male: genotype, p=0.003, substrate, p=0.0007, interaction, p=0.002.; female: genotype, ns, substrate, ns). Soleus Cmah−/− fibers from male and female mice revealed an increase conductance above WT fiber bundles that was also greater in the presence of BCAAs but only for male mice (male: genotype, p=0.003, substrate, p=0.05.; female: genotype, p=0.009, substrate, ns). Maximal respiration was increased only in male Cmah−/− soleus fibers independent of substrate. (male: genotype, p=0.002, substrate, ns.; female: genotype, ns, substrate, ns). Summary: Fibers from both the FDB and soleus of male Cmah−/− mice have a greater capacity for mitochondrial respiration under a metabolically active state that is similar to myofibers stimulated to repeatedly contraction in a hypoxic environment. Conclusion: These data suggest that effcient regulation of mitochondrial respiration utilizing BCAAs as a substrate, particularly in males, could contribute to an elite endurance phenotype. This project was funded through the UC San Diego Academic Senate Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Putative malignant hyperthermia mutation CaV1.1-R174W is insufficient to trigger a fulminant response to halothane or confer heat stress intolerance

Malignant hyperthermia susceptibility (MHS) is an autosomal dominant pharmacogenetic disorder that manifests as a hypermetabolic state when carriers are exposed to halogenated volatile anesthetics or depolarizing muscle relaxants. In animals, heat stress intolerance is also observed. MHS is linked to over 40 variants in RYR1 that are classified as pathogenic for diagnostic purposes. More recently, a few rare variants linked to the MHS phenotype have been reported in CACNA1S, which encodes the voltage-activated Ca2+ channel CaV1.1 that conformationally couples to RyR1 in skeletal muscle. Here, we describe a knock-in mouse line that expresses one of these putative variants, CaV1.1-R174W. Heterozygous (HET) and homozygous (HOM) CaV1.1-R174W mice survive to adulthood without overt phenotype but fail to trigger with fulminant malignant hyperthermia when exposed to halothane or moderate heat stress. All three genotypes (WT, HET, and HOM) express similar levels of CaV1.1 by quantitative PCR, Western blot, [3H]PN200-110 receptor binding and immobilization-resistant charge movement densities in flexor digitorum brevis fibers. Although HOM fibers have negligible CaV1.1 current amplitudes, HET fibers have similar amplitudes to WT, suggesting a preferential accumulation of the CaV1.1-WT protein at triad junctions in HET animals. Never-the-less both HET and HOM have slightly elevated resting free Ca2+ and Na+ measured with double barreled microelectrode in vastus lateralis that is disproportional to upregulation of transient receptor potential canonical (TRPC) 3 and TRPC6 in skeletal muscle. CaV1.1-R174W and upregulation of TRPC3/6 alone are insufficient to trigger fulminant malignant hyperthermia response to halothane and/or heat stress in HET and HOM mice.

Increased tetanic calcium in early fatigue of mammalian muscle fibers is accompanied by accelerated force development despite a decreased force.

During the initial phase of fatigue induced by repeated contractions in fast-twitch muscle fibers, tetanic force decreases despite increasing tetanic free cytosolic [Ca2+ ] ([Ca2+ ]cyt ). Here, we hypothesized that the increase in tetanic [Ca2+ ]cyt nevertheless has positive effects on force in early fatigue. Experiments on enzymatically isolated mouse flexor digitorum brevis (FDB) fibers showed that an increase in tetanic [Ca2+ ]cyt during ten 350 ms contractions required trains of electrical pulses to be elicited at short intervals (≤2 s) and at high frequencies (≥70 Hz). Mechanically dissected mouse FDB fibers showed greater decrease in tetanic force when the stimulation frequency during contractions was gradually reduced to prevent the increase in tetanic [Ca2+ ]cyt . Novel analyses of data from previous studies revealed an increased rate of force development in the tenth fatiguing contraction in mouse FDB fibers, as well as in rat FDB and human intercostal fibers. Mouse FDB fibers deficient in creatine kinase showed no increase in tetanic [Ca2+ ]cyt and slowed force development in the tenth contraction; after injection of creatine kinase to enable phosphocreatine breakdown, these fibers showed an increase in tetanic [Ca2+ ]cyt and accelerated force development. Mouse FDB fibers exposed to ten short contractions (43 ms) produced at short intervals (142 ms) showed increased tetanic [Ca2+ ]cyt accompanied by a marked (~16%) increase in the developed force. In conclusion, the increase in tetanic [Ca2+ ]cyt in early fatigue is accompanied by accelerated force development, which under some circumstances can counteract the decline in physical performance caused by the concomitant decrease in maximum force.

Postdevelopmental knockout of Orai1 improves muscle pathology in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD), an X-linked disorder caused by loss-of-function mutations in the dystrophin gene, is characterized by progressive muscle degeneration and weakness. Enhanced store-operated Ca2+ entry (SOCE), a Ca2+ influx mechanism coordinated by STIM1 sensors of luminal Ca2+ within the sarcoplasmic reticulum (SR) and Ca2+-permeable Orai1 channels in the sarcolemma, is proposed to contribute to Ca2+-mediated muscle damage in DMD. To directly determine the impact of Orai1-dependent SOCE on the dystrophic phenotype, we crossed mdx mice with tamoxifen-inducible, muscle-specific Orai1 knockout mice (mdx-Orai1 KO mice). Both constitutive and SOCE were significantly increased in flexor digitorum brevis fibers from mdx mice, while SOCE was absent in fibers from both Orai1 KO and mdx-Orai1 KO mice. Compared with WT mice, fibers from mdx mice exhibited (1) increased resting myoplasmic Ca2+ levels, (2) reduced total releasable Ca2+ store content, and (3) a prolonged rate of electrically evoked Ca2+ transient decay. These effects were partially normalized in fibers from mdx-Orai1 KO mice. Intact extensor digitorum longus muscles from mdx mice exhibited a significant reduction of maximal specific force, which was rescued in muscles from mdx-Orai1 KO mice. Finally, during exposure to consecutive eccentric contractions, muscles from mdx mice displayed a more pronounced decline in specific force compared with that of WT mice, which was also significantly attenuated by Orai1 ablation. Together, these results indicate that enhanced Orai1-dependent SOCE exacerbates the dystrophic phenotype and that Orai1 deficiency improves muscle pathology by both normalizing Ca2+ homeostasis and promoting sarcolemmal integrity/stability.

Mitochondrial Permeability Transition Causes Mitochondrial Reactive Oxygen Species- and Caspase 3-Dependent Atrophy of Single Adult Mouse Skeletal Muscle Fibers.

Elevated mitochondrial reactive oxygen species (mROS) and an increase in caspase-3 activity are established mechanisms that lead to skeletal muscle atrophy via the upregulation of protein degradation pathways. However, the mechanisms upstream of an increase in mROS and caspase-3 activity in conditions of muscle atrophy have not been identified. Based upon knowledge that an event known as mitochondrial permeability transition (MPT) causes an increase in mROS emission and the activation of caspase-3 via mitochondrial release of cytochrome c, as well as the circumstantial evidence for MPT in some muscle atrophy conditions, we tested MPT as a mechanism of atrophy. Briefly, treating cultured single mouse flexor digitorum brevis (FDB) fibers from adult mice with a chemical inducer of MPT (Bz423) for 24 h caused an increase in mROS and caspase-3 activity that was accompanied by a reduction in muscle fiber diameter that was able to be prevented by inhibitors of MPT, mROS, or caspase-3 (p < 0.05). Similarly, a four-day single fiber culture as a model of disuse caused atrophy that could be prevented by inhibitors of MPT, mROS, or activated caspase-3. As such, our results identify MPT as a novel mechanism of skeletal muscle atrophy that operates through mROS emission and caspase-3 activation.

Consequences of SUR2[A478V] Mutation in Skeletal Muscle of Murine Model of Cantu Syndrome.

(1) Background: Cantu syndrome (CS) arises from gain-of-function (GOF) mutations in the ABCC9 and KCNJ8 genes, which encode ATP-sensitive K+ (KATP) channel subunits SUR2 and Kir6.1, respectively. Most CS patients have mutations in SUR2, the major component of skeletal muscle KATP, but the consequences of SUR2 GOF in skeletal muscle are unknown. (2) Methods: We performed in vivo and ex vivo characterization of skeletal muscle in heterozygous SUR2[A478V] (SUR2wt/AV) and homozygous SUR2[A478V] (SUR2AV/AV) CS mice. (3) Results: In SUR2wt/AV and SUR2AV/AV mice, forelimb strength and diaphragm amplitude movement were reduced; muscle echodensity was enhanced. KATP channel currents recorded in Flexor digitorum brevis fibers showed reduced MgATP-sensitivity in SUR2wt/AV, dramatically so in SUR2AV/AV mice; IC50 for MgATP inhibition of KATP currents were 1.9 ± 0.5 × 10−5 M in SUR2wt/AV and 8.6 ± 0.4 × 10−6 M in WT mice and was not measurable in SUR2AV/AV. A slight rightward shift of sensitivity to inhibition by glibenclamide was detected in SUR2AV/AV mice. Histopathological and qPCR analysis revealed atrophy of soleus and tibialis anterior muscles and up-regulation of atrogin-1 and MuRF1 mRNA in CS mice. (4) Conclusions: SUR2[A478V] “knock-in” mutation in mice impairs KATP channel modulation by MgATP, markedly so in SUR2AV/AV, with atrophy and non-inflammatory edema in different skeletal muscle phenotypes.

Calibration of mammalian skeletal muscle Ca2+ transients recorded with the fast Ca2+ dye Mag-Fluo-4

BackgroundMag-Fluo-4 is increasingly employed for studying Ca2+ signaling in skeletal muscle; however, the lack of information on the Ca2+-Mag-Fluo-4 reaction limits its wider usage. MethodsFluorescence and isothermal titration calorimetry (ITC) experiments were performed to determine the binding stoichiometry (n) and thermodynamics (enthalpy (ΔH) and entropy (ΔS) changes), as well as the in vitro and in situ Kd of the Ca2+-Mag-Fluo-4 reaction. Rate constants (kon, koff), fluorescence maximum (Fmax), minimum (Fmin), and the dye compartmentalization were also estimated. Experiments in cells used enzymatically dissociated flexor digitorum brevis fibres of C57BL6, adult mice, loaded at room temperature for 8 min, with 6 μM Mag-Fluo-4, AM, and permeabilized with saponin or ionomycin. All measurements were done at 20 °C. ResultsThe in vitro fluorescence assays showed a binding stoichiometry of 0.5 for the Ca2+/Mag-Fluo-4 (n = 5) reaction. ITC results (n = 3) provided ΔH and ΔS values of 2.3 (0.7) kJ/mol and 97.8 (5.9) J/mol.K, respectively. The in situ Kd was 1.652 × 105μM2(n = 58 fibres, R2 = 0.99). With an Fmax of 150.9 (8.8) A.U. (n = 8), Fmin of 0.14 (0.1) A.U. (n = 10), and ΔF of Ca2+ transients of 8.4 (2.5) A.U. (n = 10), the sarcoplasmic [Ca2+]peak reached 22.5 (7.8) μM. Compartmentalized dye amounted to only 1.1 (0.7)% (n = 10).CONCLUSIONS: Two Mag-Fluo-4 molecules coalesce around one Ca2+ ion, in an entropy-driven, very low in situ affinity reaction, making it suitable to reliably track the kinetics of rapid muscle Ca2+ transients. General significanceOur results may be relevant to the quantitative study of Ca2+ kinetics in many other cell types.

A new set of equations for the simplified calibration of fluorescence Ca2+ transients in skeletal muscle fibers.

The classical approach for calibrating non-ratiometric fluorescent Ca2+ dyes entails the measurement of the fluorescence maximum (Fmax) and minimum (Fmin), as well as the dissociation constant (Kd) of the Ca2+-Dye reaction (model 1). An alternative equation does not need the Fmin but requires the rate constants kon and koff (model 2). However, both approaches are experimentally time consuming and the rate constants for several dyes are unknown. Here, we propose a set of equations (model 3) that simplify the calibration of fluorescent Ca2+ transients obtained with non-ratiometric dyes. This equation allows the calibration of signals without using the Fmin: [Ca2+] = Kd(F - Frest/Fmax - F) + [Ca2+]IR(Fmax - Frest/Fmax - F), where [Ca2+]IR is the resting [Ca2+]. If the classical calibration approach is followed, the Fmin can be estimated from: Fmin = Frest - ([Ca2+]IR(Fmax - Frest)/Kd). We tested the models' performance using signals obtained from enzymatically dissociated flexor digitorum brevis fibers of C57BL/6 mice loaded with Fluo-4, AM. Model 3 performed the same as model 2, and both gave peak [Ca2+] values 15 ± 0.3% (n = 3) lower than model 1, when we used our experimental Fmin (1.24 ± 0.11 A.U., n = 4). However, when we used the mathematically estimated Fmin (6.78 ± 0.2 A.U) for model 1, the peak [Ca2+] were similar for all three models. This suggests that the dye leakage makes a correct determination of the Fmin unlikely and induces errors in the estimation of [Ca2+]. In conclusion, we propose simpler and time-saving equations that help to reliably calibrate cytosolic Ca2+ transients obtained with non-ratiometric fluorescent dyes. The use of the estimated Fmin avoids the uncertainties associated with its experimental measurement.

Senescence Is Associated With Elevated Intracellular Resting [Ca2 +] in Mice Skeletal Muscle Fibers. An in vivo Study.

Aging causes skeletal muscles to become atrophied, weak, and easily fatigued. Here, we have tested the hypothesis that normal aging in skeletal muscle cells is associated with Ca2+ intracellular dyshomeostasis and oxidative stress. Intracellular Ca2+ concentration ([Ca2+]i), resting intracellular Na+ concentration ([Na+]i) and reactive oxygen species (ROS) production were measured in vivo (superficial gastrocnemius fibers) using double-barreled ion-selective microelectrodes, and in vitro [isolated single flexor digitorum brevis fibers] using fluorescent ROS sensor CM-H2DCFDA in young (3 months of age), middle-aged (12 months of age), and aged (24 months of age) mice. We found an age-related increase in [Ca2+]i from 121 ± 4 nM in young muscle cells which rose to 255 ± 36 nM in middle-aged and to 409 ± 25 nM in aged cells. [Na+]i also showed an age-dependent elevation, increasing from 8 ± 0.5 mM in young muscle fibers, to 12 ± 1 mM in middle-aged and to 17 ± 1 mM in old muscle fibers. Using the fluorescent ROS sensor CM-H2DCFDA we found that these increases in intracellular cation concentrations were associated with significantly increased basal ROS production as demonstrated by age related increases in the rate of dichlorodihydrofluorescein fluorescence. To determine is this could be modified by reducing ROS and/or blocking sarcolemmal Ca2+ influx we administered flufenamic acid (FFA), a non-steroidal anti-inflammatory drug which is also a non-selective blocker of the transient receptor potential canonical channels (TRPCs), for 4 weeks to determine if this would have a beneficial effect. FFA treatment reduced both basal ROS production and muscle [Ca2+]i and [Na+]i in middle-aged and aged muscle fibers compared to fibers and muscles of untreated 12 and 24-months old mice. [Ca2+]i was reduced to 134 ± 8 nM in middle-aged muscle and to 246 ± 40 nM in muscle from aged mice. Likewise [Na+]i was reduced to 9 ± 0.7 mM in middle-aged muscles and to 13 ± 1 mM in muscle from aged mice. FFA treatment also reduced age associated increases in plasma interleukin 6 and tumor necrosis factor-alpha (TNF-α) concentrations which were elevated in 12 and 24-months old mice compared to young mice and decreased age-related muscle damage as indicated by a reduction in serum creatine kinase (CK) activity. Our data provides a direct demonstration that normal aging is associated with a significant elevation [Ca2+]i, [Na+]i, and intracellular ROS production in skeletal muscle fibers. Furthermore, the fact that FFA reduced the intracellular [Ca2+], [Na+], and ROS production as well as the elevated IL6, TNF-α, and CK levels, led us to suggest that its pharmacological effect may be related to its action both as a TRPC channel blocker and as an anti-inflammatory.

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.

Biophysical and Pharmacological Characterization of Atp-Sensitive Potassium Channels in Mice Kir6.1WT/V65M Mirroring the Human Cantu′ Syndrome

Cantu syndrome(CS) arises from GOF mutations in KCNJ8 or ABCC9, the genes encoding for Kir6.1 and SUR2 subunits of ATP-sensitive potassium(KATP) channels. Kir6.1 is prominent in vascular and skeletal muscle, but absent from cardiac muscle. Heterozygous Kir6.1[V65M] mice, in which the CS mutation was knocked in to the endogenous locus by CRISPR/Cas9 gene editing, exhibit KATP over-activity and reduced contractility in vascular smooth muscle, but not cardiomyocite. No information is available for other tissues. We measured the forelimb strength and ultrasonography of diaphragm and skeletal muscle, and investigated the KATP channel properties in native flexor digitorum brevis (FDB) and soleus (SOL) fibers, skin fibroblasts and long bones/calvaria-derived osteoblasts from WT and Kir6.1wt/V65M mice, by patch clamp technique. Forelimb forces were lower, and ultrasonography evaluation of the hind-limb showed significant enhancement of echodensity, in Kir6.1wt/V65M mice with respect to WT. Increased organ/tissue weights were observed in Kir6.1wt/V65M mice with respect to WT. In patch clamp experiments, higher KATP amplitude, reflecting the GOF associated with the mutation was observed in FDB fibers, skin fibroblasts and long bones/calvaria- derived osteoblasts from Kir6.1wt/V65M mice with respect to WT. No differences in KATP amplitude were detected between WT vs Kir6.1wt/V65M SOL fibers. A marked loss of channel sensitivity to Glib, also characteristic of the mutation was observed in all the mutant cells vs controls. Finally, a lower FDB fiber survival was observed in Kir6.1wt/V65M mice respect to the WT mice within 24 hours of isolation. The CS mutation Kir6.1wt/V65M has significant effects on KATP currents in multiple skeletal muscle and other tissues, reducing skeletal muscle contractility and fiber viability. This suggests that skeletal muscle insufficiency could be a significant issue in CS patients suffering from Kir6.1 GOF mutations.

Astaxanthin Improves Tetanic Force Without Altering Skeletal Muscle Excitation-Contraction Coupling in Mice

Astaxanthin (AX) is a powerful natural anti-oxidant that protects against the risk of oxidative damage from free radicals and reactive oxygen species. Up to date, there are no studies concerning how and if AX feeding affects the excitation-contraction coupling (ECC) mechanism in skeletal muscle. 8 young adult C57Bl6 mice were divided into two groups: 1) control and 2) AX supplemented for 4 weeks (AstaReal1010, 0.05% w/w added in standard rodent chow). Following the feeding the AX group gained significantly less weight on average (0.16±0.33g vs 1.22±0.31g). When examining the in vitro force of the extensor digitorum longus (EDL) muscle the amplitude of single twitches (5 V, 100 ms) was not altered, but the amplitude of tetani (200 Hz, 200 ms) was significantly increased in the AX group. We also measured the activity-dependent mitochondrial Ca2+ uptake in rhod-2 [SzP1] loaded flexor digitorum brevis (FDB) fibers at rest and during tetanic stimulation. The average (±SE) time course of mitochondrial normalized fluorescence was significantly decreased in the AX group at rest (0.28±0.01 vs 0.12±0.01), after the 1st tetanus (0.3±0.01 vs 0.13±0.01) and after the 5th tetanus (0.39±0.08 vs 0.14±0.02) (p<0.05), respectively. Lastly, we studied the voltage activation of calcium transients in single FDB fibers under whole cell voltage clamp. The voltage dependence of calcium release (fitted with the Boltzmann function) was not statistically different between the two groups (V1/2: -12.4±2.3 mV, k=6.7±0.5 for Ctrl, n=12 vs. V1/2: -14.9±2.7 mV, k=8.1±0.8 for AX, n=17). We conclude that AX supplementation increases the in vitro force of the EDL muscles without affecting the ECC mechanism in skeletal muscle. AX feeding seem to exert a protecting effect on the mitochondria as the Ca2+ uptake was significantly less in the AX group.

Adenylosuccinic acid therapy ameliorates murine Duchenne Muscular Dystrophy

Arising from the ablation of the cytoskeletal protein dystrophin, Duchenne Muscular Dystrophy (DMD) is a debilitating and fatal skeletal muscle wasting disease underpinned by metabolic insufficiency. The inability to facilitate adequate energy production may impede calcium (Ca2+) buffering within, and the regenerative capacity of, dystrophic muscle. Therefore, increasing the metabogenic potential could represent an effective treatment avenue. The aim of our study was to determine the efficacy of adenylosuccinic acid (ASA), a purine nucleotide cycle metabolite, to stimulate metabolism and buffer skeletal muscle damage in the mdx mouse model of DMD. Dystrophin-positive control (C57BL/10) and dystrophin-deficient mdx mice were treated with ASA (3000 µg.mL−1) in drinking water. Following the 8-week treatment period, metabolism, mitochondrial density, viability and superoxide (O2−) production, as well as skeletal muscle histopathology, were assessed. ASA treatment significantly improved the histopathological features of murine DMD by reducing damage area, the number of centronucleated fibres, lipid accumulation, connective tissue infiltration and Ca2+ content of mdx tibialis anterior. These effects were independent of upregulated utrophin expression in the tibialis anterior. ASA treatment also increased mitochondrial viability in mdx flexor digitorum brevis fibres and concomitantly reduced O2− production, an effect that was also observed in cultured immortalised human DMD myoblasts. Our data indicates that ASA has a protective effect on mdx skeletal muscles.

Measurement of Skeletal Muscle Fiber Contractility with High-Speed Traction Microscopy

We describe a technique for simultaneous quantification of the contractile forces and cytosolic calcium dynamics of muscle fibers embedded in three-dimensional biopolymer gels under auxotonic loading conditions. We derive a scaling law for linear elastic matrices such as basement membrane extract hydrogels (Matrigel) that allows us to measure contractile force from the shape of the relaxed and contracted muscle cell and the Young’s modulus of the matrix without further knowledge of the matrix deformations surrounding the cell and without performing computationally intensive inverse force reconstruction algorithms. We apply our method to isolated mouse flexor digitorum brevis (FDB) fibers that are embedded in 10 mg/mL Matrigel. Upon electrical stimulation, individual FDB fibers show twitch forces of 0.37 ± 0.15 μN and tetanic forces (100-Hz stimulation frequency) of 2.38 ± 0.71 μN, corresponding to a tension of 0.44 ± 0.25 kPa and 2.53 ± 1.17 kPa, respectively. Contractile forces of FDB fibers increase in response to caffeine and the troponin-calcium stabilizer tirasemtiv, similar to responses measured in whole muscle. From simultaneous high-speed measurements of cell length changes and cytosolic calcium concentration using confocal line scanning at a frequency of 2048 Hz, we show that twitch and tetanic force responses to electric pulses follow the low-pass filtered calcium signal. In summary, we present a technically simple high-speed method for measuring contractile forces and cytosolic calcium dynamics of single muscle fibers. We expect that our method will help to reduce preparation time, costs, and the number of sacrificed animals needed for experiments such as drug testing.

Equivalent L-type channel (CaV1.1) function in adult female and male mouse skeletal muscle fibers

Loss of total muscle force during aging has both atrophic and non-atrophic components. The former deficit is a direct consequence of reduced muscle mass while the latter has been attributed to a depression of excitation-contraction (EC) coupling. It is well established that age-onset reductions in sex hormone production regulate the atrophic component in both males and females. However, it is unknown whether the non-atrophic component is influenced by sex hormones. Since the non-atrophic component has been linked mechanistically to reduced expression of the skeletal muscle L-type Ca2+ channel (CaV1.1), we recorded L-type Ca2+ currents, gating charge movements and depolarization-induced changes in myoplasmic Ca2+ from flexor digitorum brevis (FDB) fibers of naïve and gonadectomized mice of both sexes. Our first set of experiments sought to identify any basal differences in EC coupling or L-type Ca2+ flux between the sexes; no detectable differences in any of the aforementioned parameters were observed between FDB harvested from either naïve males or females. In the latter segments of the study, ovariectomy (OVX) and orchiectomy (ORX) models were used to assess the possible influence of sex hormones on EC coupling and/or L-type Ca2+ flux. In these experiments, FDB fibers harvested from OVX and ORX mice both showed no differences in L-type Ca2+ current, gating charge movement or depolarization-induced changes in Ca2+ release from the sarcoplasmic reticulum. Taken together, our results indicate L-type Ca2+ channel function and EC coupling are: 1) equivalent between the sexes, and 2) not significantly regulated by sex hormones. Since recent NIH review guidelines mandate the consideration of sex differences as a criterion for review, our work indicates the suitability of either sex for the study of the fundamental mechanisms of EC coupling. Thus, our findings may accelerate the research process by conserving animals, labor and financial resources.

Moderately elevated extracellular [K+] potentiates submaximal force and power in skeletal muscle via increased [Ca2+]i during contractions.

The extracellular K+ concentration ([K+]o) increases during physical exercise. We here studied whether moderately elevated [K+]o may increase force and power output during contractions at in vivo-like subtetanic frequencies and whether such potentiation was associated with increased cytosolic free Ca2+ concentration ([Ca2+]i) during contractions. Isolated whole soleus and extensor digitorum longus (EDL) rat muscles were incubated at different levels of [K+]o, and isometric and dynamic contractility were tested at various stimulation frequencies. Furthermore, [Ca2+]i at rest and during contraction was measured along with isometric force in single mouse flexor digitorum brevis (FDB) fibers exposed to elevated [K+]o. Elevating [K+]o from 4 mM up to 8 mM (soleus) and 11 mM (EDL) increased isometric force at subtetanic frequencies, 2-15 Hz in soleus and up to 50 Hz in EDL, while inhibition was seen at tetanic frequency in both muscle types. Elevating [K+]o also increased peak power of dynamic subtetanic contractions, with potentiation being more pronounced in EDL than in soleus muscles. The force-potentiating effect of elevated [K+]o was transient in FDB single fibers, reaching peak after ~4 and 2.5 min in 9 and 11 mM [K+]o, respectively. At the time of peak potentiation, force and [Ca2+]i during 15-Hz contractions were significantly increased, whereas force was slightly decreased and [Ca2+]i unchanged during 50-Hz contractions. Moderate elevation of [K+]o can transiently potentiate force and power during contractions at subtetanic frequencies, which can be explained by a higher [Ca2+]i during contractions.

Electroporated GLUT4-7myc-GFP detects in vivo glucose transporter 4 translocation in skeletal muscle without discernible changes in GFP patterns.

What is the central question of this study? Resolving the mechanism(s) leading to glucose transporter 4 (GLUT4) translocation to the muscle surface membrane has great therapeutic potential. However, the measurement of GLUT4 translocation is technically challenging. Here, we asked whether electroporation of GLUT4-7myc-GFP into skeletal muscle could be used as a tool to study GLUT4 translocation in vivo. What is the main finding and its importance? By acutely inducing GLUT4-7myc-GFP expression in skeletal muscle, we verified that in vivo exercise and AICAR stimulation increased the GLUT4 presence in the sarcolemma measured as myc signal. Importantly, the increased myc signal in the sarcolemma was not accompanied by major visual changes in the distribution of the GFP signal. Insulin and exercise lead to translocation of the glucose transporter4 (GLUT4) to the surface membrane of skeletal muscle fibres. This process is pivotal for facilitating glucose uptake into skeletal muscle. To study this, a robust assay is needed to measure the translocation of GLUT4 in adult skeletal muscle directly. Here, we aimed to validate a simple GLUT4 translocation assay using a genetically encoded biosensor in mouse skeletal muscle. We transfected GLUT4-7myc-GFP into mouse muscle to study live GLUT4 movement and to evaluate GLUT4 insertion in the muscle surface membrane after in vivo running exercise and pharmacological activation of AMP-activated protein kinase (AMPK). Transfection led to expression of GLUT4-7myc-GFP that was dynamic in live flexor digitorum brevis fibres and which, upon insulin stimulation, exposed the myc epitope extracellularly. Running exercise, in addition to AMPK activation by 5-aminoimidazole-4-carboxamide ribonucleotide, induced ∼125 and ∼100% increase, respectively, in extracellularly exposure of GLUT4 in the surface membrane of tibialis anterior muscle. Interestingly, the clear increase in surface-exposed GLUT4 content induced by insulin, exercise or AMPK activation was not accompanied by any discernible reorganization of the GLUT4-GFP signal. In conclusion, we provide a detailed description of an easy-to-use translocation assay to study GLUT4 accumulation at the surface membrane induced by exercise and exercise-mimicking stimuli. Notably, our analyses revealed that increased GLUT4 surface membrane accumulation was not accompanied by a discernible change in the GLUT4 localization pattern.

Defects in sarcolemma repair and skeletal muscle function after injury in a mouse model of Niemann-Pick type A/B disease

BackgroundNiemann-Pick disease type A (NPDA), a disease caused by mutations in acid sphingomyelinase (ASM), involves severe neurodegeneration and early death. Intracellular lipid accumulation and plasma membrane alterations are implicated in the pathology. ASM is also linked to the mechanism of plasma membrane repair, so we investigated the impact of ASM deficiency in skeletal muscle, a tissue that undergoes frequent cycles of injury and repair in vivo.MethodsUtilizing the NPDA/B mouse model ASM−/− and wild type (WT) littermates, we performed excitation-contraction coupling/Ca2+ mobilization and sarcolemma injury/repair assays with isolated flexor digitorum brevis fibers, proteomic analyses with quadriceps femoris, flexor digitorum brevis, and tibialis posterior muscle and in vivo tests of the contractile force (maximal isometric torque) of the quadriceps femoris muscle before and after eccentric contraction-induced muscle injury.ResultsASM−/− flexor digitorum brevis fibers showed impaired excitation-contraction coupling compared to WT, a defect expressed as reduced tetanic [Ca2+]i in response to electrical stimulation and early failure in sustaining [Ca2+]i during repeated tetanic contractions. When injured mechanically by needle passage, ASM−/− flexor digitorum brevis fibers showed susceptibility to injury similar to WT, but a reduced ability to reseal the sarcolemma. Proteomic analyses revealed changes in a small group of skeletal muscle proteins as a consequence of ASM deficiency, with downregulation of calsequestrin occurring in the three different muscles analyzed. In vivo, the loss in maximal isometric torque of WT quadriceps femoris was similar immediately after and 2 min after injury. The loss in ASM−/− mice immediately after injury was similar to WT, but was markedly larger at 2 min after injury.ConclusionsSkeletal muscle fibers from ASM−/− mice have an impairment in intracellular Ca2+ handling that results in reduced Ca2+ mobilization and a more rapid decline in peak Ca2+ transients during repeated contraction-relaxation cycles. Isolated fibers show reduced ability to repair damage to the sarcolemma, and this is associated with an exaggerated deficit in force during recovery from an in vivo eccentric contraction-induced muscle injury. Our findings uncover the possibility that skeletal muscle functional defects may play a role in the pathology of NPDA/B disease.

\u03b2-arrestin 1 regulates \u03b22-adrenergic receptor-mediated skeletal muscle hypertrophy and contractility

Backgroundβ2-adrenergic receptors (β2ARs) are the target of catecholamines and play fundamental roles in cardiovascular, pulmonary, and skeletal muscle physiology. An important action of β2AR stimulation on skeletal muscle is anabolic growth, which has led to the use of agonists such as clenbuterol by athletes to enhance muscle performance. While previous work has demonstrated that β2ARs can engage distinct signaling and functional cascades mediated by either G proteins or the multifunctional adaptor protein, β-arrestin, the precise role of β-arrestin in skeletal muscle physiology is not known. Here, we tested the hypothesis that agonist activation of the β2AR by clenbuterol would engage β-arrestin as a key transducer of anabolic skeletal muscle growth.MethodsThe contractile force of isolated extensor digitorum longus muscle (EDL) and calcium signaling in isolated flexor digitorum brevis (FDB) fibers were examined from the wild-type (WT) and β-arrestin 1 knockout mice (βarr1KO) followed by chronic administration of clenbuterol (1 mg/kg/d). Hypertrophic responses including fiber composition and fiber size were examined by immunohistochemical imaging. We performed a targeted phosphoproteomic analysis on clenbuterol stimulated primary cultured myoblasts from WT and βarr1KO mice. Statistical significance was determined by using a two-way analysis with Sidak’s or Tukey’s multiple comparison test and the Student’s t test.ResultsChronic administration of clenbuterol to WT mice enhanced the contractile force of EDL muscle and calcium signaling in isolated FDB fibers. In contrast, when administered to βarr1KO mice, the effect of clenbuterol on contractile force and calcium influx was blunted. While clenbuterol-induced hypertrophic responses were observed in WT mice, this response was abrogated in mice lacking β-arrestin 1. In primary cultured myoblasts, clenbuterol-stimulated phosphorylation of multiple pro-hypertrophy proteins required the presence of β-arrestin 1.ConclusionsWe have identified a previously unappreciated role for β-arrestin 1 in mediating β2AR-stimulated skeletal muscle growth and strength. We propose these findings could have important implications in the design of future pharmacologic agents aimed at reversing pathological conditions associated with skeletal muscle wasting.

Optical Recording of Action Potential Initiation and Propagation in Mouse Skeletal Muscle Fibers

Skeletal muscle fibers have been used to examine a variety of cellular functions and pathologies. Among other parameters, skeletal muscle action potential (AP) propagation has been measured to assess the integrity and function of skeletal muscle. In this work, we utilize 1-(3-sulfonatopropyl)-4[β[2-(Di-n-octylamino)-6-naphtyl]vinyl]pyridinium betaine, a potentiometric dye, and mag-fluo-4, a low-affinity intracellular Ca2+indicator, to noninvasively and reliably measure AP conduction velocity in skeletal muscle. We used remote extracellular bipolar electrodes to generate an alternating polarity electric field that initiates an AP at either end of the fiber. Using enzymatically dissociated flexor digitorum brevis (FDB) fibers and high-speed line scans, we determine the conduction velocity to be ∼0.4 m/s. We applied these methodologies to FDB fibers under elevated extracellular potassium conditions and confirmed that the conduction velocity is significantly reduced in elevated [K+]o. Because our recorded velocities for FDB fibers were much slower than previously reported for other muscle groups, we compared the conduction velocity in FDB fibers to that of extensor digitorum longus (EDL) fibers and measured a significantly faster velocity in EDL fibers than FDB fibers. As a basis for this difference in conduction velocity, we found a similarly higher level of expression of Na+ channels in EDL than in FDB fibers. In addition to measuring the conduction velocity, we can also measure the passive electrotonic potentials elicited by pulses by applying tetrodotoxin and have constructed a circuit model of a skeletal muscle fiber to predict passive polarization of the fiber by the field stimuli. Our predictions from the model fiber closely resemble the recordings acquired from in vitro assays. With these techniques, we can examine how various pathologies and mutations affect skeletal muscle AP propagation. Our work demonstrates the utility of using 1-(3-sulfonatopropyl)-4[β[2-(Di-n-octylamino)-6-naphtyl]vinyl]pyridinium betaine or mag-fluo-4 to noninvasively measure AP initiation and conduction.