Skeletal Muscle Contraction Research Articles

The Relationship Between Length and Active Force for Submaximal Skeletal Muscle Contractions: a Review.

The force-length relationship is usually obtained for isometric contractions with maximal activation, but less is known about how sarcomere length affects force during submaximal activation. During submaximal activation, length-dependent alterations in calcium sensitivity, owing to changes in cross-bridge kinetics (rate of attachment and/or detachment), result in an activation-dependent shift in optimal length to longer sarcomere lengths. It is known that sarcomere length, as well as temperature and phosphorylation of the regulatory light chains of myosin, can modify Ca2⁺ sensitivity by altering the probability of cross-bridge interaction. This altered calcium sensitivity is particularly important for submaximal force levels, as it can change the shape of the length dependence of force, with peak force occurring at sarcomere lengths longer than those associated with maximal filament overlap. In athletic contexts, contractions typically do not reach maximal intensity. Therefore, understanding that the ability to produce force under both maximal and submaximal conditions can differ, and that peak force can be generated at different lengths, could influence the development of targeted training regimens optimal for each sport.

Effect of suspension training intervention on the balance ability of juvenile deaf-mute patients

Maintaining balance is essential for daily activities; deficits in hearing-impaired adolescents can severely impact their academics and daily life. Suspension training is employed to enhance the coordination of multiple muscle groups within the body and ultimately to improve balance. A total of 105 students with non-genetic hearing impairments, comprising 65 boys and 40 girls, participated in the study. Balance training using the TRS suspension belt included exercises such as prone elbow brace, supine back brace, kneeling elbow brace, lateral elbow brace, prone hip flexion, and supine hip lift. Strength training using equipment involved exercises like single-leg suspension squat, flying bird brace, supine suspension arm flexion, seated static brace, and standing butterfly pinch chest. The main outcome measures included static balance tests (lambda footwork, Wolfson postural stress test), dynamic balance tests (functional forward reach test, balance beam walk test), and vestibule function tests (rotation-walk test). The results of the lambda footwork and Wolfson postural stress tests revealed highly significant differences between 8 weeks and 16 weeks of intervention (p < 0.01). The Functional Forward Stretch test demonstrated a significant difference between 8 weeks of intervention and 2 weeks of intervention at 16 weeks (p < 0.05). The balance beam walking test indicated a significant improvement at 8 weeks (p < 0.05) and a highly significant improvement at 16 weeks (p < 0.01). In the spin-walk test, no statistically significant differences were observed between the pre-experimental test, the 8-week intervention, and the 16-week intervention (p > 0.05). Leveraging the expropriation system to mediate skeletal muscle movement and muscle contraction coordination can improve balance. Suspension training significantly improved balance during activities requiring static non-interference maintenance. However, suspension training did not demonstrate a significant effect on improving vestibule function. Overall, suspension training was effective in enhancing balance in all patients with non-hereditary deafness.

Stable oxidative posttranslational modifications alter the gating properties of RyR1.

The ryanodine receptor type 1 (RyR1) is a Ca2+ release channel that regulates skeletal muscle contraction by controlling Ca2+ release from the sarcoplasmic reticulum (SR). Posttranslational modifications (PTMs) of RyR1, such as phosphorylation, S-nitrosylation, and carbonylation are known to increase RyR1 open probability (Po), contributing to SR Ca2+ leak and skeletal muscle dysfunction. PTMs on RyR1 have been linked to muscle dysfunction in diseases like breast cancer, rheumatoid arthritis, Duchenne muscle dystrophy, and aging. While reactive oxygen species (ROS) and oxidative stress induce PTMs, the impact of stable oxidative modifications like 3-nitrotyrosine (3-NT) and malondialdehyde adducts (MDA) on RyR1 gating remains unclear. Mass spectrometry and single-channel recordings were used to study how 3-NT and MDA modify RyR1 and affect Po. Both modifications increased Po in a dose-dependent manner, with mass spectrometry identifying 30 modified residues out of 5035 amino acids per RyR1 monomer. Key modifications were found in domains critical for protein interaction and channel activation, including Y808/3NT in SPRY1, Y1081/3NT and H1254/MDA in SPRY2&3, and Q2107/MDA and Y2128/3NT in JSol, near the binding site of FKBP12. Though these modifications did not directly overlap with FKBP12 binding residues, they promoted FKBP12 dissociation from RyR1. These findings provide detailed insights into how stable oxidative PTMs on RyR1 residues alter channel gating, advancing our understanding of RyR1-mediated Ca2+ release in conditions associated with oxidative stress and muscle weakness.

Muscle RING-finger proteins (MuRF) regulate protein kinase A activity via retrograde vesicular transport of PKA RI-alpha

Abstract Background Muscle RING finger (MuRF) proteins are muscle-restricted E3 ubiquitin ligases that control protein degradation in striated muscles. MuRF proteins have coordinate functions for maintenance of striated muscle structure and function that depends on their target proteins. MuRF1 is a key enzyme in muscle atrophy. MuRF2 and MuRF3 bind to and stabilize microtubules which affects striated muscle differentiation and contraction. Although some MuRF-interaction partners have been described only few have been proven to be targeted for proteasomal degradation. To better understand the function of MuRF proteins, we set out to identify and functionally characterize novel MuRF target proteins. Methods and Results Stable isotope labeling of amino acids in cell culture followed by affinity purification and quantitative mass spectrometry analysis (SILAC-AP-MS) was used to identify the interactome of MuRF proteins. We identified the mammalian retromer subunit sorting nexin 5 (SNX5) that is involved in subcellular trafficking as a novel MuRF2 and MuRF3 interaction partner. Coimmunoprecipitation experiments and immunocytochemistry confirmed physical interaction and colocalization between SNX5 and MuRF2 or MuRF3. The interaction domains were mapped. MuRF2, MuRF3 and SNX5 were colocalized to early endosomes at microtubules in myocytes. MuRF2 mediated the ubiquitin proteasome system-dependent degradation of SNX5 in a RING-finger dependent manner, whereas MuRF3 stabilized SNX5. Using mass spectrometry, we identified the regulatory subunit of protein kinase A (PKA-RI-α) as a cargo of SNX5 coated early endosomes in myocytes. CRISPR-Cas9 and siRNA experiments showed that SNX5 regulates PKA activity by stabilizing PKA-RI-α in myocytes. Deletion of SNX5 resulted in PKA activation and inhibition of the HDAC5-MEF2 axis that disturbed myogenic differentiation. Conclusion MuRF2 and MuRF3 play opposing roles in SNX5-mediated retrograde transport of early endosomes along microtubules in myocytes. This mechanism is involved in regulation of PKA catalytic activity via stabilization of PKA-RI-α and controls differentiation of striated muscles.

7138 Testosterone Modulation of Muscle Transcriptome Profile During Intensive Lifestyle Intervention in Older Men With Obesity and Hypogonadism

Abstract Disclosure: V. Viola: None. T. Samanta: None. M. Duremdes Nava: None. A. Celli: None. R.C. Villareal: None. N.H. Nguyen: None. G. Colleluori: None. Y. Barnouin: None. N. Napoli: None. C. Qualls: None. B.A. Kaipparettu: None. D.T. Villareal: None. Background: We reported that testosterone replacement added to lifestyle therapy can mitigate weight loss-induced reduction of muscle mass and bone mineral density (BMD) in older men with obesity and hypogonadism (Barnouin et al., J Clin Endocrinol Metab, 2021). Objective: To investigate the molecular mechanisms underlying the mitigation of muscle and BMD loss in response to testosterone replacement therapy during intensive lifestyle intervention in this at-risk population. Materials and Methods: Among the 83 older (≥ 65 y) men with obesity (BMI ≥ 30 kg/m2) and hypogonadism (early AM testosterone persistently <300 ng/dl) associated with frailty (PPT score ≤ 31) who were randomized into 26-week lifestyle therapy plus testosterone (LT+Test) or placebo (LT+Pbo), 38 underwent serial muscle biopsies for the muscle transcriptomics substudy. Results: Despite a similar 9% weight loss, lean body mass and thigh muscle volume decreased less in LT+Test than LT+Pbo (both -2% vs -4%, respectively; p=0.04). Hip BMD was preserved in LT+Test compared with LT+Pbo (0.4% vs -1.1%; p=0.03). Muscle strength increased similarly in LT+Test and LT+Pbo (23% vs 24%; p=0.95). Total testosterone increased more in LT+Test than LT+Pbo (133% vs 32%; p=0.01). Based on Next Generation Sequencing, of the 39,160 and 39,115 genes detected in LT+Test and LT+Pbo, respectively, 151 genes were differentially expressed (DEGs) in LT+Test and 114 in LT+Pbo group. Gene Ontology enrichment analyses revealed that in the LT+Test group, just four muscle-related terms (muscle organ development, muscle organ morphogenesis, regulation of skeletal muscle contraction, muscle atrophy) were downregulated, and one term (muscle system process) was upregulated while in the LT+Pbo, nine terms related to muscle (muscle system process, muscle tissue development, muscle organ development, skeletal muscle tissue development, skeletal muscle organ development, skeletal muscle cell differentiation, muscle organ morphogenesis, response to stimuli involved in regulation of muscle adaptation, muscle atrophy), and one term related to bone (bone mineralization involved in bone maturation) were downregulated. Notably, muscle system process was upregulated in the LT+Test but downregulated in the LT+Pbo. The RT-PCR analyses showed that LT+Test resulted in a higher expression of MYOD1 (p=0.02) and WNT4 (p= 0.08), key genes involved in muscle and bone metabolism, respectively, compared to LT+Pbo. Furthermore, we observed significantly higher mRNA expression of MYBPH (p=0.01), SCN3B (p=0.02), and DSC2 (p=0.01), genes involved in the muscle system process, in response to LT+Test compared to LT+Pbo. Conclusion: The addition of testosterone replacement to lifestyle therapy mitigates the weight loss-induced reduction of muscle mass and BMD via countering the weight loss-induced downregulation of genes involved in muscle and bone anabolism. Presentation: 6/2/2024

Influence of native thin filament type on the regulation of atrial and ventricular myosin motor activity

Ca2+-mediated activation of thin filaments is a crucial step in initiating striated muscle contraction. To gain mechanistic insight into this regulatory process, thin filament (TF) components and myosin motors from diverse species and tissue sources are often combined in minimal in vitro systems. The contribution of tissue-specific TF composition with native myosin motors in generating contraction speed remains unclear. To examine TF-mediated regulation, we established a procedure to purify native TFs (nTF) and myosin motors (M-II) from the same cardiac tissue samples as low as 10 mg and investigated their influence on gliding speeds and Ca2+ sensitivity. The rabbit atrial and ventricular nTFs and M-II were assessed in in vitro nTF motility experiments under varying Ca2+ concentrations. The speed-pCa relationship yielded a maximum TF speed of 2.58 μm/s for atrial (aM-II) and 1.51 μm/s for ventricular myosin (vM-II), both higher than the respective unregulated actin filament gliding speeds. The Ca2+ sensitivity was different for both protein sources. After swapping the nTFs, the ventricular TFs increased their gliding speed on atrial myosin, while the atrial nTFs reduced their gliding speed on ventricular myosin. Swapping of the nTFs decreased the calcium sensitivity for both vM-II and aM-II, indicating a strong influence of the thin filament source. These studies suggest that the nTF-myosin combination is critical to understanding the Ca2+ sensitivity of the shortening speed. Our approach is highly relevant to studying precious human cardiac samples, i.e., small myectomy samples, to address the alteration of contraction speed and Ca2+ sensitivity in cardiomyopathies.

Changes in masseter muscle structure, membrane lipid peroxidation and Ca-ATPase activity as effects of different local anesthetics.

Local anesthetics (LA) can cause undesired effects such as sustained contraction of skeletal muscles as a result of structural and functional changes. Proper skeletal muscle function is controlled by intracellular Ca2+ concentration and efficient energy (ATP) production, which is closely related to cell ultrastructure. Aim: The aim of this study was to identify the structural and functional changes caused by LAs. Materials and Method: Male Wistar rats weighing 200 to 250g were used (n:49). They were divided into seven groups. One group was not anesthetized or treated (Control). The other six groups underwent intramuscular (IM) anesthesia with xylazine 2% (0.05 ml) and ketamine 50 mg/ml (0.1 ml/100g rat weight), and one of the following was applied to the masseter muscle (MM): no further treatment (Anesthetic Control group, CA); 0.1ml physiological saline solution (group SF); Carrageenin (group Carr) 1% as positive control group; prilocaine (group Pri), mepivacaine (group Mepi); or articaine (group Arti) 0.3M, IM. The animals were euthanized by cervical dislocation one hour after treatment. The effects of the different anesthetics on the MM were evaluated histologically and by electronic microscopy (EM). Ca-ATPase and membrane lipid peroxidation (LPX) were evaluated in muscle homogenates under the same conditions as those used to prepare the histological sections. Results: In general, structural damage and increased muscle contraction were observed in tissues treated with anesthetics. The most extreme values of Ca-ATPase activity and LPX were observed in the positive control group (carrageenin). Results were analyzed by one-way ANOVA for multiple comparisons and Tukey's test (p < 0.05). Conclusions: The results suggest that in the short term, local anesthetics affect the muscle function and are associated to structural changes.

Myokines May Be the Answer to the Beneficial Immunomodulation of Tailored Exercise-A Narrative Review.

Exercise can regulate the immune function, activate the activity of immune cells, and promote the health of the organism, but the mechanism is not clear. Skeletal muscle is a secretory organ that secretes bioactive substances known as myokines. Exercise promotes skeletal muscle contraction and the expression of myokines including irisin, IL-6, BDNF, etc. Here, we review nine myokines that are regulated by exercise. These myokines have been shown to be associated with immune responses and to regulate the proliferation, differentiation, and maturation of immune cells and enhance their function, thereby serving to improve the health of the organism. The aim of this article is to review the effects of myokines on intrinsic and adaptive immunity and the important role that exercise plays in them. It provides a theoretical basis for exercise to promote health and provides a potential mechanism for the correlation between muscle factor expression and immunity, as well as the involvement of exercise in body immunity. It also provides the possibility to find a suitable exercise training program for immune system diseases.

Dual role of the S5 segment in type 1 ryanodine receptor channel gating

The type 1 ryanodine receptor (RyR1) is a Ca2+ release channel in the sarcoplasmic reticulum that is essential for skeletal muscle contraction. RyR1 forms a channel with six transmembrane segments, in which S5 is the fifth segment and is thought to contribute to pore formation. However, its role in channel gating remains unclear. Here, we performed a functional analysis of several disease-associated mutations in S5 and interpreted the results with respect to the published RyR1 structures to identify potential interactions associated with the mutant phenotypes. We demonstrate that S5 plays a dual role in channel gating: the cytoplasmic side interacts with S6 to reduce the channel activity, whereas the luminal side forms a rigid structural base necessary for S6 displacement in channel opening. These results deepen our understanding of the molecular mechanisms of RyR1 channel gating and provide insight into the divergent disease phenotypes caused by mutations in S5.

Protein kinase C epsilon contributes to chronic mechanoreflex sensitization in rats with heart failure.

We investigated second-messenger signalling components linked to the stimulation of Gq protein-coupled receptors (e.g. thromboxane A2 and bradykinin B2 receptors) on the sensory endings of thin fibre muscle afferents in the chronic mechanoreflex sensitization in rats with myocardial infarction-induced heart failure with reduced ejection fraction (HF-rEF). We hypothesized that injection of either the inositol 1,4,5-trisphosphate (IP3) receptor antagonist xestospongin C (5µg) or the PKCε translocation inhibitor PKCe141 (45µg) into the arterial supply of the hindlimb would reduce the increase in renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) evoked during 30s of 1Hz dynamic hindlimb muscle stretch in decerebrate, unanaesthetized HF-rEF rats but not sham-operated controls (SHAM). Ejection fraction was significantly reduced in HF-rEF (45 (19)%) compared to SHAM (80 (9)%; P<0.001) rats. In HF-rEF rats (n=3M/2F), IP3 receptor blockade had no effect on the peak ΔRSNA (pre: 99 (74)%; post: 133 (79)%; P=0.974) or peak ΔMAP response to stretch (peak ΔMAP: pre: 32 (14) mmHg; post: 36 (21) mmHg; P=0.719). Conversely, in another group of HF-rEF rats (n=4M/3F), the PKCε translocation inhibitor reduced the peak ΔRSNA (pre: 110 (77)%; post: 62 (58)%; P=0.029) and peak ΔMAP response to stretch (pre: 30 (20) mmHg; post: 17 (16) mmHg; P=0.048). In SHAM counterparts, neither drug affected the mechanoreflex responses. Our findings highlight PKCε, but not IP3 receptors, as a significant second-messenger in the chronic mechanoreflex sensitization in HF-rEF which may play a crucial role in the exaggerated sympathetic response to exercise in this patient population. KEY POINTS: Skeletal muscle contraction results in an exaggerated reflex increase in sympathetic nerve activity in heart failure patients with reduced ejection fraction (HF-rEF) compared to healthy individuals, contributing to increased cardiovascular risk and impaired tolerance for mild exercise. The exaggerated reflex sympathetic responses in HF-rEF may be attributed to a chronic sensitization of mechanically sensitive thin fibre muscle afferents mediated, at least in part, by stimulation of Gq protein-coupled thromboxane A2 and bradykinin B2 receptors on muscle afferent sensory endings. The specific Gq protein-linked signalling mechanisms that produce the chronic mechanoreflex sensitization in HF-rEF have not been investigated but may involve inositol 1,4,5-trisphosphate (IP3) receptors and/or protein kinase C epsilon (PKCε). Here we demonstrate that PKCε, but not IP3 receptors, within the sensory endings of thin fibre muscle afferents plays a role in the sensitization of mechanically sensitive thin fibre muscle afferents in rats with HF-rEF.

Abstract 58: The 5-HT7 receptor contributes to increased hindquarter blood flow caused by skeletal muscle contraction

Exogenous serotonin (5-hydroxytryptamine, 5-HT) causes a reproducible decrease in blood pressure in normotensive and hypertensive rats. This is associated with dilation of arterioles in skeletal muscle. The 5-HT7 receptor KO rat strain allowed us to test the role of this receptor in both responses as well as the effects of endogenous 5-HT -- acting at 5-HT7 receptors -- on normal circulatory regulation. First, we hypothesized that the 5-HT7 receptor KO rat would have a higher hindquarter (HQ) vascular resistance (HQVR) than wildtype (WT) because of loss of this receptor. In rats anesthetized with isoflurane, instrumented with arterial catheters and flow probes on the terminal aorta, male KO rats had significantly higher resting HQVR [mm Hg/ml/min; KO (16.0±2.0) vs WT (10.8±0.6.0), p = 0.04] and lower HQ blood flow than normal Sprague-Dawley (SD) rats. No differences in resting HQVR or HQ flow were observed in female rats. Intravenous infusion of 5-HT (50 ug/kg/min) reduced systemic blood pressure and HQVR; these parameters were significantly attenuated (~56%) in male KO rats versus WT and SD animals. In females, falls in systemic blood pressure and HQVR were absent in both KO and WT rats. Second, as 5-HT has been implicated in the pathophysiology of heart failure (HF), a left anterior descending artery ligation model of HF was used to investigate the potential role of 5-HT7 regulation of HQ flow in HF-associated exercise intolerance. The ability of hindlimb muscle stimulation to increase blood flow using transdermal neuromuscular electrical stimulation was tested (NMES). M and F KO rats showed a reduced ability to increase HQ flow during muscle contraction vs WT (% over basal: M WT = 118±18 vs KO =14.6±7.1; F WT= 101±12 vs KO = 7.6±6) rats. This was observed in both intact and HF rats (in which flow changes were already impaired). Acute administration of the 5-HT7 receptor antagonist SB269970 abolished the ability of NMES to increase HQ flow in normal SD rats. Importantly, the microcirculatory capacity, visualized by Lectin-594, was not significantly different in the gastrocnemius muscle of WT (arbitrary fluorescence units/area: 946±40) vs KO (914±69). These data support that the 5-HT7 receptor, likely activated by endogenous 5-HT, is an important regulator of skeletal muscle vascular resistance. Thus, 5-HT may have a larger role in normal and pathophysiological regulation of the circulation than has previously been appreciated.

Changes in masseter muscle structure, membrane lipid peroxidation and Ca-ATPase activity as effects of different local anesthetics

Local anesthetics (LA) can cause undesired effects such as sustained contraction of skeletal muscles as a result of structural and functional changes. Proper skeletal muscle function is controlled by intracellular Ca2+ concentration and efficient energy (ATP) production, which is closely related to cell ultrastructure. Aim: The aim of this study was to identify the structural and functional changes caused by LAs. Materials and Method: Male Wistar rats weighing 200 to 250g were used (n:49). They were divided into seven groups. One group was not anesthetized or treated (Control). The other six groups underwent intramuscular (IM) anesthesia with xylazine 2% (0.05 ml) and ketamine 50 mg/ ml (0.1 ml/100g rat weight), and one of the following was applied to the masseter muscle (MM): no further treatment (Anesthetic Control group, CA); 0.1ml physiological saline solution (group SF); Carrageenin (group Carr) 1% as positive control group; prilocaine (group Pri), mepivacaine (group Mepi); or articaine (group Arti) 0.3M, IM. The animals were euthanized by cervical dislocation one hour after treatment. The effects of the different anesthetics on the MM were evaluated histologically and by electronic microscopy (EM). Ca-ATPase and membrane lipid peroxidation (LPX) were evaluated in muscle homogenates under the same conditions as those used to prepare the histological sections. Results: In general, structural damage and increased muscle contraction were observed in tissues treated with anesthetics. The most extreme values of Ca-ATPase activity and LPX were observed in the positive control group (carrageenin). Results were analyzed by one-way ANOVA for multiple comparisons and Tukey’s test (p &lt; 0.05). Conclusions: The results suggest that in the short term, local anesthetics affect the muscle function and are associated to structural changes.

CaV1.1 voltage-sensing domain III exclusively controls skeletal muscle excitation-contraction coupling

Skeletal muscle contractions are initiated by action potentials, which are sensed by the voltage-gated calcium channel (CaV1.1) and are conformationally coupled to calcium release from intracellular stores. Notably, CaV1.1 contains four separate voltage-sensing domains (VSDs), which activate channel gating and excitation-contraction (EC-) coupling at different voltages and with distinct kinetics. Here we show that a single VSD of CaV1.1 controls skeletal muscle EC-coupling. Whereas mutations in VSDs I, II and IV affect the current properties but not EC-coupling, only mutations in VSD III alter the voltage-dependence of depolarization-induced calcium release. Molecular dynamics simulations reveal comprehensive, non-canonical state transitions of VSD III in response to membrane depolarization. Identifying the voltage sensor that activates EC-coupling and detecting its unique conformational changes opens the door to unraveling the downstream events linking VSD III motion to the opening of the calcium release channel, and thus resolving the signal transduction mechanism of skeletal muscle EC-coupling.

Identification of human metabolites of fast skeletal troponin activators Tirasemtiv and Reldesemtiv for doping control purposes.

The fast skeletal troponin activators (FSTAs) Reldesemtiv and Tirasemtiv were developed for patients suffering from neuro-degenerative diseases of the motor nervous system, e.g. amyotrophic lateral sclerosis (ALS). The drug candidates can increase the sensitivity of troponin C to calcium by selectively activating the troponin complex resulting in increased skeletal muscle contraction. Although the development of the drug candidates is currently discontinued because of missed end points in phase III clinical studies with patients with ALS, phase I clinical trials showed an increase in muscle contraction force in healthy humans. This effect could be abused by athletes to enhance performance in sports. As the substances are listed on the 2024 edition of the World Anti-Doping Agency's Prohibited List, the aim of this study was to identify and characterize metabolites of Reldesemtiv and Tirasemtiv to ensure their reliable identification in doping control analyses. The biotransformation of the drug candidates was studied in vitro using pooled human liver microsomes and 3D cultivated human hepatic cells of the cell line HepaRG, yielding a total of 11 metabolites of Reldesemtiv and eight of Tirasemtiv. In addition, a human elimination study was conducted to investigate the metabolism and elimination profile of Tirasemtiv and Reldesemtiv in vivo, suggesting the N-glucuronide of Tirasemtiv and hydroxylated 3-fluoro-2-(3-fluoro-1-methylcyclobutyl)pyridine as well as its glucuronide as suitable target analytes for routine doping controls. Applying a validating HPLC-MS/MS method, optimized to detect Reldesemtiv and Tirasemtiv in human urine, microdosing (50 μg) of each substance was traceable for 24-72 h.

Abstract Mo121: Sarcomere activation biosensor reveals key functional differences in live cell active states between cardiac and skeletal muscle

The sarcomere is the functional unit of striated muscle contraction. Previous studies have provided valuable information about the sarcomeric proteins and modulators that are necessary for activation of contraction through inter-myofilament signaling. This work has revealed key differences in contraction kinetics and paradigms between cardiac, slow skeletal, and fast skeletal muscle. We seek here to expand these studies through the use of myofilament incorporated Förster Resonance Energy Transfer (FRET) fluorescent biosensor in live, intact muscle. This biosensor fuses fluorescent donor and acceptor proteins to the N- and C-terminals of fast skeletal and cardiac (slow) troponin C, respectively, and allows for the monitoring in real-time of the stepwise physiological mechanism of muscle sarcomere activation in an intact system which preserves significant features of the muscle, including excitation-contraction coupling and load. This biosensor has been designed and validated in both cardiac and skeletal muscle and using intact muscles from transgenic animals we have demonstrated FRET fluorescence in cardiac papillaries, extensor digitorum longus, and soleus muscles. This biosensor has shown unique sarcomere activation properties between cardiac and skeletal muscle, notably the emergence of a prolonged return to baseline of the FRET signal in loaded fast and slow skeletal muscle, which is evidence of a “primed state” of myofilament activation unique to skeletal muscles under load. Elucidation of the “primed state” of sarcomere activation is evidence of a transient memory of recent activity enabling the enhanced contraction properties of skeletal muscle. This finding advances previous understanding of skeletal muscle activation, including summation and tetanic contraction, which is not possible in cardiac muscle. The implementation of this biosensor provides a new approach to interrogate the essential physiological features of muscle sarcomere activation and contraction and adds a valuable new tool for small molecule and gene-based discoveries for muscle disease remediation.

A case of opioid-induced rigidity requiring naloxone administration at the time of anesthesia emergence

BackgroundOpioid-induced rigidity is typically observed during rapid administration of fentanyl. Herein, we present a case in which rigidity occurred after reversal of rocuronium during emergence from anesthesia.Case presentationA 73-year-old man underwent video-assisted partial lung resection. General anesthesia was induced with propofol, remimazolam, remifentanil, and rocuronium. Fentanyl was administered early during anesthesia. The surgery was completed without complications, and sugammadex sodium was administered for rocuronium reversal. The patient became agitated, but spontaneous breathing was maintained; therefore, the intratracheal tube was removed after the administration of flumazenil. The patient developed stiffness in the neck and jaw muscles along with remarkable skeletal muscle contractions. Dramatic improvement was observed immediately after administration of naloxone.ConclusionsEven as the simulated effect site concentration of fentanyl decreases during anesthesia emergence, opioid-induced rigidity may still occur. Rapid reversal of remimazolam by flumazenil might have contributed to the rigidity in this case.

Structural switch in acetylcholine receptors in developing muscle.

During development, motor neurons originating in the brainstem and spinal cord form elaborate synapses with skeletal muscle fibres1. These neurons release acetylcholine (ACh), which binds to nicotinic ACh receptors (AChRs) on the muscle, initiating contraction. Two types of AChR are present in developing muscle cells, and their differential expression serves as a hallmark of neuromuscular synapse maturation2-4. The structural principles underlying the switch from fetal to adult muscle receptors are unknown. Here, we present high-resolution structures of both fetal and adult muscle nicotinic AChRs, isolated from bovine skeletal muscle in developmental transition. These structures, obtained in the absence and presence of ACh, provide a structural context for understanding how fetal versus adult receptor isoforms are tuned for synapse development versus the all-or-none signalling required for high-fidelity skeletal muscle contraction. We find that ACh affinity differences are driven by binding site access, channel conductance is tuned by widespread surface electrostatics and open duration changes result from intrasubunit interactions and structural flexibility. The structures further reveal pathogenic mechanisms underlying congenital myasthenic syndromes.

Effects of membrane cholesterol-targeting chemicals on skeletal muscle contractions evoked by direct and indirect stimulation.

Cholesterol is one of the major components of plasma membrane, where its distribution is nonhomogeneous and it participates in lipid raft formation. In skeletal muscle cholesterol and lipid rafts seem to be important for excitation-contraction coupling and for neuromuscular transmission, involving cholesterol-rich synaptic vesicles. In the present study, nerve and muscle stimulation-evoked contractions were recorded to assess the role of cholesterol in contractile function of mouse diaphragm. Exposure to cholesterol oxidase (0.2 U/ml) and cholesterol-depleting agent methyl-β-cyclodextrin (1 mM) did not affect markedly contractile responses to both direct and indirect stimulation at low and high frequency. However, methyl-β-cyclodextrin at high concentration (10 mM) strongly decreased the force of both single and tetanus contractions induced by phrenic nerve stimulation. This decline in contractile function was more profoundly expressed when methyl-β-cyclodextrin application was combined with phrenic nerve activation. At the same time, 10 mM methyl-β-cyclodextrin had no effect on contractions upon direct muscle stimulation at low and high frequency. Thus, strong cholesterol depletion suppresses contractile function mainly due to disturbance of the neuromuscular communication, whereas muscle fiber contractility remains resistant to decline.

Kinetics and mapping of Ca-driven calmodulin conformations on skeletal and cardiac muscle ryanodine receptors

Calmodulin transduces [Ca2+] information regulating the rhythmic Ca2+ cycling between the sarcoplasmic reticulum and cytoplasm during contraction and relaxation in cardiac and skeletal muscle. However, the structural dynamics by which calmodulin modulates the sarcoplasmic reticulum Ca2+ release channel, the ryanodine receptor, at physiologically relevant [Ca2+] is unknown. Using fluorescence lifetime FRET, we resolve different structural states of calmodulin and Ca2+-driven shifts in the conformation of calmodulin bound to ryanodine receptor. Skeletal and cardiac ryanodine receptor isoforms show different calmodulin-ryanodine receptor conformations, as well as binding and structural kinetics with 0.2-ms resolution, which reflect different functional roles of calmodulin. These FRET methods provide insight into the physiological calmodulin-ryanodine receptor structural states, revealing additional distinct structural states that complement cryo-EM models that are based on less physiological conditions. This technology will drive future studies on pathological calmodulin-ryanodine receptor interactions and dynamics with other important ryanodine receptor bound modulators.

Electrical Pulse Stimulation Protects C2C12 Myotubes against Hydrogen Peroxide-Induced Cytotoxicity via Nrf2/Antioxidant Pathway.

Skeletal muscle contraction evokes numerous biochemical alterations that underpin exercise benefits. This present study aimed to elucidate the mechanism for electrical pulse stimulation (EPS)-induced antioxidant adaptation in C2C12 myotubes. We found that EPS significantly upregulated Nrf2 and a broad array of downstream antioxidant enzymes involved in multiple antioxidant systems. These effects were completely abolished by pretreatment with a ROS scavenger, N-acetylcysteine. MitoSOX-Red, CM-H2DCFDA, and EPR spectroscopy revealed a significantly higher ROS level in mitochondria and cytosol in EPS cells compared to non-stimulated cells. Seahorse and Oroboros revealed that EPS significantly increased the maximal mitochondrial oxygen consumption rate, along with an upregulated protein expression of mitochondrial complexes I/V, mitofusin-1, and mitochondrial fission factor. A post-stimulation time-course experiment demonstrated that upregulated NQO1 and GSTA2 last at least 24 h following the cessation of EPS, whereas elevated ROS declines immediately. These findings suggest an antioxidant preconditioning effect in the EPS cells. A cell viability study suggested that the EPS cells displayed 11- and 36-fold higher survival rates compared to the control cells in response to 2 and 4 mM H2O2 treatment, respectively. In summary, we found that EPS upregulated a large group of antioxidant enzymes in C2C12 myotubes via a contraction-mitochondrial-ROS-Nrf2 pathway. This antioxidant adaptation protects cells against oxidative stress-associated cytotoxicity.