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.

Structural basis of the super- and hyper-relaxed states of myosin II

Super-relaxation is a state of muscle thick filaments in which ATP turnover by myosin is much slower than that of myosin II in solution. This inhibited state, in equilibrium with a faster (relaxed) state, is ubiquitous and thought to be fundamental to muscle function, acting as a mechanism for switching off energy-consuming myosin motors when they are not being used. The structural basis of super-relaxation is usually taken to be a motif formed by myosin in which the two heads interact with each other and with the proximal tail forming an interacting-heads motif, which switches the heads off. However, recent studies show that even isolated myosin heads can exhibit this slow rate. Here, we review the role of head interactions in creating the super-relaxed state and show how increased numbers of interactions in thick filaments underlie the high levels of super-relaxation found in intact muscle. We suggest how a third, even more inhibited, state of myosin (a hyper-relaxed state) seen in certain species results from additional interactions involving the heads. We speculate on the relationship between animal lifestyle and level of super-relaxation in different species and on the mechanism of formation of the super-relaxed state. We also review how super-relaxed thick filaments are activated and how the super-relaxed state is modulated in healthy and diseased muscles.

Can Tumbling without Brine Improve Tenderness and Proteolysis of Beef Loin Muscles?

Tumbling of intact muscle foods has been widely applied toprocessed meats using brine solution. However, the use of tumbling withoutbrine on fresh beef muscles has not been fully examined. Therefore, this studyaimed to evaluate fresh beef tumbling on meat quality and proteolytic featuresof loin (longissimus lumborum)muscles. Moreover, interactions with the duration of postmortem aging wereinvestigated. Loins (n=9) at 7d postmortem were sectioned and allocated among twotumbling (T) treatment groups at 60 (T60) or 90 (T90) minutes, as well as a non-tumbledcontrol (T0) group. After treatment, sub-sections were made and divided among0d, 7d, or 14d of further aging. Meat quality was assessed by shear forcevalues, water-holding ability, and color attributes. The extent of proteolysiswas determined by quantification of desmin and troponin-T, myofibrilfragmentation index (MFI), and transmission electron microscopy. An interactionbetween fresh beef tumbling and aging duration was observed in shear forcevalues (P=0.032). At 0d, muscles fromT90 exhibited lower shear force (21.6 N) compared to T0 (34.8 N) and T60 (24.7N) groups. Muscles from T60 and T90 groups maintained lower shear force than T0controls at each respective aging duration.Higher cooking loss (P=0.011) but notpurge loss (P=0.412) was observed in theT60 and T90 groups compared to T0. Shear force results were supported by higherMFI in T60 and T90 groups than T0 controls (P<0.001), as well as the disappearance of intact troponin-T withfurther aging (P=0.009). Transmissionelectron microscopy supported increased initial tenderness would owe primarily tophysical disruptions to myofibrillar structure, though fresh beef tumbling may facilitateproteolysis with further aging.

Cyclophosphamide treatment evoked side effect on skeletal muscle actin, monitored by DSC

Several kind of drugs—used in cancer treatments—such as cyclophosphamide (CP) can also trigger a disease classified as toxic polyneuropathy. Polyneuropathy is a simultaneous malfunction of several peripheral nerves, typical side effect of a cancer therapy. In our previous study, we used CP treated in vitro animal model (Guinea pig) with a comparable dosage and time handling of human protocol to show evidences of this drug-induced effects. We could show a dose-dependent difference between in Tm and ΔHcal of untreated and treated samples assigned to their intact muscle and nerve, blood plasma and red blood cells. In our current study we analyze this side effect on skeletal muscle actin (prepared from m. psoas of rabbit) by DSC (differential scanning calorimetry), to follow the possible consequence of drug treatment on the “activator” of muscle contraction. We have demonstrated that run of DSC curves, Tms together with the ΔHcal exhibit clear CP effect. In case of Ca2+ G actin it is manifested in a well separated second high denaturing temperature as a consequence of CP binding into the cleft. This way the nucleotide binding cleft with subdomains 1 and 3 becomes less flexible, indicating clear sensitivity to CP treatment. In F-actin samples, the main peak represents the thermal denaturation of subdomains 1 and 3, and the increased calorimetric enthalpy administrating Ca2+ as well as CP refers to a more rigid structure. These alterations can be the molecular background in the malfunction of muscle in case of polyneuropathy after CP treatment.

Detection of Ca2+ transients near ryanodine receptors by targeting fluorescent Ca2+ sensors to the triad

In intact muscle fibers, functional properties of ryanodine receptor (RYR)–mediated sarcoplasmic reticulum (SR) Ca2+ release triggered by activation of the voltage sensor CaV1.1 have so far essentially been addressed with diffusible Ca2+-sensitive dyes. Here, we used a domain (T306) of the protein triadin to target the Ca2+-sensitive probe GCaMP6f to the junctional SR membrane, in the immediate vicinity of RYR channels, within the triad region. Fluorescence of untargeted GCaMP6f was distributed throughout the muscle fibers and experienced large Ca2+-dependent changes, with obvious kinetic delays, upon application of voltage-clamp depolarizing pulses. Conversely, T306-GCaMP6f localized to the triad and generated Ca2+-dependent fluorescence transients of lower amplitude and faster kinetics for low and intermediate levels of Ca2+ release than those of untargeted GCaMP6f. By contrast, model simulation of the spatial gradients of Ca2+ following Ca2+ release predicted limited kinetic differences under the assumptions that the two probes were present at the same concentration and suffered from identical kinetic limitations. At the spatial level, T306-GCaMP6f transients within distinct regions of a same fiber yielded a uniform time course, even at low levels of Ca2+ release activation. Similar observations were made using GCaMP6f fused to the γ1 auxiliary subunit of CaV1.1. Despite the probe's limitations, our results point out the remarkable synchronicity of voltage-dependent Ca2+ release activation and termination among individual triads and highlight the potential of the approach to visualize activation or closure of single groups of RYR channels. We anticipate targeting of improved Ca2+ sensors to the triad will provide illuminating insights into physiological normal RYR function and its dysfunction under stress or pathological conditions.

Unique Metabolomic Profile of Skeletal Muscle in Chronic Limb Threatening Ischemia

Chronic limb threatening ischemia (CLTI) is the most severe manifestation of peripheral atherosclerosis. Patients with CLTI have poor muscle quality and function and are at high risk for limb amputation and death. The objective of this study was to interrogate the metabolome of limb muscle from CLTI patients. To accomplish this, a prospective cohort of CLTI patients undergoing either a surgical intervention (CLTI Pre-surgery) or limb amputation (CLTI Amputation), as well as non-peripheral arterial disease (non-PAD) controls were enrolled. Gastrocnemius muscle biopsy specimens were obtained and processed for nuclear magnetic resonance (NMR)-based metabolomics analyses using solution state NMR on extracted aqueous and organic phases and 1H high-resolution magic angle spinning (HR-MAS) on intact muscle specimens. CLTI Amputation specimens displayed classical features of ischemic/hypoxic metabolism including accumulation of succinate, fumarate, lactate, alanine, and a significant decrease in the pyruvate/lactate ratio. CLTI Amputation muscle also featured aberrant amino acid metabolism marked by elevated branched chain amino acids. Finally, both Pre-surgery and Amputation CLTI muscles exhibited pronounced accumulation of lipids, suggesting the presence of myosteatosis, including cholesterol, triglycerides, and saturated fatty acids. Taken together, these metabolite differences add to a growing body of literature that have characterized profound metabolic disturbance’s in the failing ischemic limb of CLTI patients.

Dependence of thick filament structure in relaxed mammalian skeletal muscle on temperature and interfilament spacing

Contraction of skeletal muscle is regulated by structural changes in both actin-containing thin filaments and myosin-containing thick filaments, but myosin-based regulation is unlikely to be preserved after thick filament isolation, and its structural basis remains poorly characterized. Here, we describe the periodic features of the thick filament structure in situ by high-resolution small-angle x-ray diffraction and interference. We used both relaxed demembranated fibers and resting intact muscle preparations to assess whether thick filament regulation is preserved in demembranated fibers, which have been widely used for previous studies. We show that the thick filaments in both preparations exhibit two closely spaced axial periodicities, 43.1 nm and 45.5 nm, at near-physiological temperature. The shorter periodicity matches that of the myosin helix, and x-ray interference between the two arrays of myosin in the bipolar filament shows that all zones of the filament follow this periodicity. The 45.5-nm repeat has no helical component and originates from myosin layers closer to the filament midpoint associated with the titin super-repeat in that region. Cooling relaxed or resting muscle, which partially mimics the effects of calcium activation on thick filament structure, disrupts the helical order of the myosin motors, and they move out from the filament backbone. Compression of the filament lattice of demembranated fibers by 5% Dextran, which restores interfilament spacing to that in intact muscle, stabilizes the higher-temperature structure. The axial periodicity of the filament backbone increases on cooling, but in lattice-compressed fibers the periodicity of the myosin heads does not follow the extension of the backbone. Thick filament structure in lattice-compressed demembranated fibers at near-physiological temperature is similar to that in intact resting muscle, suggesting that the native structure of the thick filament is largely preserved after demembranation in these conditions, although not in the conditions used for most previous studies with this preparation.

Effects of radiofrequency on muscle tissue regeneration

Background: Radiofrequency (RF) is recommended to treat pathologies with the presence of inflammation, as it induces diathermyand, consequently, promotes better oxygenation, nutrition and local vasodilation. Objective: Evaluate the effect of RF on muscleregeneration in Wistar rats. Methods: It is a controlled and randomized experiment, with a sample composed of 40 Wistar rats,divided equally into four groups: G1 (control group), G2 (lesion, without RF), G3 (RF after 72 hours of lesion) and G4 (RF after 7 daysof lesion), all sacrificed 21 days after the injury. The RF parameters used were: Sine wave; frequency of 0.5MHz; 5 cm² treatmentarea on the region around the lesion; power of 45%; two-minute application; intensity of 15 seconds to heat the head, 1 minute at20% and another minute at 10%. An optical microscope was used for histological analysis and, for the biomechanical properties(maximum elongation and maximum load), the mechanical traction test of the gastrocnemius muscles. For statistical analysis, thetwo-way ANOVA test and the Benferroni test were used, considering 5% of significance. Results: It was observed in G3 that theinflammatory process was optimized by the RF when compared to the other groups, presenting intact muscle fibers with a discreetregeneration process. G4, on the other hand, revealed intense inflammation with significant granulation tissue, as well as fibrosis andhealing. As for the biomechanical characteristics, there were no statistically significant differences in the intergroup comparison.Conclusion: RF was more effective when applied after 72 hours after the injury, in addition to not interfering with musclebiomechanical characteristics.

Tuning of feedforward control enables stable muscle force-length dynamics after loss of autogenic proprioceptive feedback

Animals must integrate feedforward, feedback and intrinsic mechanical control mechanisms to maintain stable locomotion. Recent studies of guinea fowl (Numida meleagris) revealed that the distal leg muscles rapidly modulate force and work output to minimize perturbations in uneven terrain. Here we probe the role of reflexes in the rapid perturbation responses of muscle by studying the effects of proprioceptive loss. We induced bilateral loss of autogenic proprioception in the lateral gastrocnemius muscle (LG) using self-reinnervation. We compared in vivo muscle dynamics and ankle kinematics in birds with reinnervated and intact LG. Reinnervated and intact LG exhibit similar steady state mechanical function and similar work modulation in response to obstacle encounters. Reinnervated LG exhibits 23ms earlier steady-state activation, consistent with feedforward tuning of activation phase to compensate for lost proprioception. Modulation of activity duration is impaired in rLG, confirming the role of reflex feedback in regulating force duration in intact muscle.