Acute exposure to extracellular BTP2 does not inhibit Ca2+ release during EC coupling in intact skeletal muscle fibers

The inhibitor of store-operated Ca2+ entry (SOCE) BTP2 was reported to inhibit ryanodine receptor Ca2+ leak and electrically evoked Ca2+ release from the sarcoplasmic reticulum when introduced into mechanically skinned muscle fibers. However, it is unclear how effects of intracellular application of a highly lipophilic drug like BTP2 on Ca2+ release during excitation–contraction (EC) coupling compare with extracellular exposure in intact muscle fibers. Here, we address this question by quantifying the effect of short- and long-term exposure to 10 and 20 µM BTP2 on the magnitude and kinetics of electrically evoked Ca2+ release in intact mouse flexor digitorum brevis muscle fibers. Our results demonstrate that neither the magnitude nor the kinetics of electrically evoked Ca2+ release evoked during repetitive electrical stimulation were altered by brief exposure (2 min) to either BTP2 concentration. However, BTP2 did reduce the magnitude of electrically evoked Ca2+ release in intact fibers when applied extracellularly for a prolonged period of time (30 min at 10 µM or 10 min at 20 µM), consistent with slow diffusion of the lipophilic drug across the plasma membrane. Together, these results indicate that the time course and impact of BTP2 on Ca2+ release during EC coupling in skeletal muscle depends strongly on whether the drug is applied intracellularly or extracellularly. Further, these results demonstrate that electrically evoked Ca2+ release in intact muscle fibers is unaltered by extracellular application of 10 µM BTP2 for <25 min, validating this use to assess the role of SOCE in the absence of an effect on EC coupling.

Preliminary Observations on Skeletal Muscle Adaptation and Plasticity in Homer 2-/- Mice

Homer represents a diversified family of scaffold and transduction proteins made up of several isoforms. Here, we present preliminary observations on skeletal muscle adaptation and plasticity in a transgenic model of Homer 2-/- mouse using a multifaceted approach entailing morphometry, quantitative RT-PCR, confocal immunofluorescence, and electrophysiology. Morphometry shows that Soleus muscle (SOL), at variance with Extensor digitorum longus muscle (EDL) and Flexor digitorum brevis muscle (FDB), displays sizable reduction of fibre cross-sectional area compared to the WT counterparts. In SOL of Homer 2-/- mice, quantitative RT-PCR indicated the upregulation of Atrogin-1 and Muscle ring finger protein 1 (MuRF1) genes, and confocal immunofluorescence showed the decrease of neuromuscular junction (NMJ) Homer content. Electrophysiological measurements of isolated FDB fibres from Homer 2-/- mice detected the exclusive presence of the adult ε-nAChR isoform excluding denervation. As for NMJ morphology, data were not conclusive, and further work is needed to ascertain whether the null Homer 2 phenotype induces any endplate remodelling. Within the context of adaptation and plasticity, the present data show that Homer 2 is a co-regulator of the normotrophic status in a muscle specific fashion.

A Huge Plantar Intramuscular Hemangioma in the Plantar Area Treated Surgically: A Case Report and Literature Review

Intramuscular hemangioma (IH) is rare, accounting for only 0.8% of all hemangioma cases. In particular, IH of the foot has only been reported a few times. In such cases, the symptoms typically include tenderness and swelling, often in relation to physical activity, but tingling or impaired function may also be present. Here, we report a patient who presented with a significant IH in the plantar area treated surgically. A 25-year-old female visited our hospital with pain in the plantar aspect of the right foot. She had noticed a mass about 10 years prior. She had previously experienced pain only when pressing the mass, but the pain subsequently became more regular pain and was exacerbated by exercise. In fact, the pain became so intense that she could not sleep well. Upon physical examination, mild swelling and tenderness of the plantar area were noted in the second to the fourth metatarsal. Sensation and motor reflexes were normal and the results of Tinel’s test were negative. Plain radiographs of the right foot revealed phleboliths scattered throughout the first to third intermetatarsal spaces. Magnetic resonance imaging revealed a space-occupying multilobulated mass (5.6 × 2.8 × 2.5 cm) located in the flexor digitorum brevis (FDB) muscle, which penetrated the plantar fascia and spread to the subcutaneous layer. In T2-weighted images, the lesion displayed a hyperintense signal compared to the surrounding skeletal muscle. Based on radiological findings, we suspected IH. The mass surrounded by the FDB muscle was exposed and completely removed via wide excision. IH consisting of cavernous-like vascular structures was diagnosed on pathology. At 1-year follow-up, the patient was almost asymptomatic and had recovered almost full range of motion in the plantar area. Histological analysis and surgery are recommended to remove intramuscular hemangiomas in the plantar area, but if the patient is not suitable for surgery, sclerotherapy or combination treatment should also be considered.

Transcriptomic analysis of mdx mouse muscles reveals a signature of early human Duchenne muscular dystrophy

The mdx mouse (C57BL/10ScSn-DMDmdx/J) is the oldest model of Duchenne muscular dystrophy (DMD). However, the mdx mouse has a nearly normal lifespan and mild pathology while DMD remains a severe, fatal disease. New mouse models that are more severely affected have not replaced the mdx mouse in DMD research. Here we report RNA-seq analysis of 55 wild-type and mdx mouse muscles: the hindlimb flexor digitorum brevis (FDB), extensor digitorum longus (EDL) and soleus (SOL) muscles, from 2- and 5-month-old mice. We investigate features of the mdx pathology and compare them with human DMD data. The mdx mouse muscles show enrichment of pathways related not only to inflammation and the immune response, but also to metabolic, developmental and structural pathways. The FDB shows a slower pathology development than EDL and SOL, in agreement with ex vivo experiments showing a similar age dependence of microtubule-related production of reactive oxygen species. Furthermore, the three mdx muscles show changes in over 80 genes affected in pre-symptomatic DMD patients. The mild pathology of the mdx mouse thus best mimics the early stages of DMD. This study should be helpful to those using mdx or mdx-derived mouse models to improve or develop DMD treatments.

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.

Single cell RNA-seq analysis of the flexor digitorum brevis mouse myofibers

Background Skeletal muscle myofibers can be separated into functionally distinct cell types that differ in gene and protein expression. Current single cell expression data is generally based upon single nucleus RNA, rather than whole myofiber material. We examined if a whole-cell flow sorting approach could be applied to perform single cell RNA-seq (scRNA-seq) in a single muscle type. Methods We performed deep, whole cell, scRNA-seq on intact and fragmented skeletal myofibers from the mouse fast-twitch flexor digitorum brevis muscle utilizing a flow-gated method of large cell isolation. We performed deep sequencing of 763 intact and fragmented myofibers. Results Quality control metrics across the different gates indicated only 171 of these cells were optimal, with a median read count of 239,252 and an average of 12,098 transcripts per cell. scRNA-seq identified three clusters of myofibers (a slow/fast 2A cluster and two fast 2X clusters). Comparison to a public skeletal nuclear RNA-seq dataset demonstrated a diversity in transcript abundance by method. RISH validated multiple genes across fast and slow twitch skeletal muscle types. Conclusion This study introduces and validates a method to isolate intact skeletal muscle myofibers to generate deep expression patterns and expands the known repertoire of fiber-type-specific genes.

Stepping onto the unknown: reflexes of the foot and ankle while stepping with perturbed perceptions of terrain

Unanticipated variations in terrain can destabilize the body. The foot is the primary interface with the ground and we know that cutaneous reflexes provide important sensory feedback. However, little is known about the contribution of stretch reflexes from the muscles within the foot to upright stability. We used intramuscular electromyography measurements of the foot muscles flexor digitorum brevis (FDB) and abductor hallucis (AH) to show for the first time how their short-latency stretch reflex response (SLR) may play an important role in responding to stepping perturbations. The SLR of FDB and AH was highest for downwards steps and lowest for upwards steps, with the response amplitude for level and compliant steps in between. When the type of terrain was unknown or unexpected to the participant, the SLR of AH and the ankle muscle soleus tended to decrease. We found significant relationships between the contact kinematics and forces of the leg and the SLR, but a person's expectation still had significant effects even after accounting for these relationships. Motor control models of short-latency body stabilization should not only include local muscle dynamics, but also predictions of terrain based on higher level information such as from vision or memory.