Prenatal murine skeletogenesis partially recovers from absent skeletal muscle as development progresses

Skeletal muscle contractions are critical for normal growth and morphogenesis of the skeleton, but it is unclear how the detrimental effects of absent muscle on the bones and joints change over time. Joint size, shape and cavitation, and rudiment length and mineralisation were assessed in multiple rudiments at two developmental stages (Theiler Stage (TS)24 and TS27) in the splotch-delayed 'muscleless limb' mouse model and littermate controls. As development progressed, the effects of absent muscle on all parameters except for cavitation become less severe. All major joints in muscleless limbs were qualitatively and quantitatively abnormal in shape at TS24, while, by TS27, most muscleless joint shapes were normal, or nearly normal. In contrast, any joints which were fused at TS24 did not cavitate by TS27. Therefore, recovery in joint shape over development occurred despite absent cavitation. Mineralisation showed the most pronounced changes between TS24 and TS27 in the muscleless limbs. At TS24, all muscleless rudiments studied had less mineralisation than the controls, while at TS27, muscleless limb rudiments had either the same or significantly more mineralisation than controls of the same age. We conclude that the effects of absent muscle on prenatal murine skeletogenesis are most pronounced in early skeletal development and reduce in severity prior to birth. Understanding how mammalian bones and joints continue to develop in an environment without muscle contractions, but with mechanical stimulation due to the movement of the mother, provides important insights into conditions affecting human babies such as developmental dysplasia of the hip and arthrogryposis.

A novel methodology for the synchronous collection and multimodal visualisation of continuous neurocardiovascular and neuromuscular physiological data in adults with long COVID

Reports suggest that adults with post-COVID-19 syndrome or long COVID may be affected by orthostatic intolerance syndromes, with autonomic nervous system dysfunction as a possible causal factor of neurocardiovascular instability (NCVI). Long COVID can also manifest as prolonged fatigue, which may be linked to neuromuscular function impairment (NMFI). The current clinical assessment for NCVI monitors neurocardiovascular performance upon the application of orthostatic stressors such as an active (i.e. self-induced) stand or a passive (tilt table) standing test. Lower limb muscle contractions may be important in orthostatic recovery via the skeletal muscle pump. In this study, adults with long COVID were assessed with a protocol that, in addition to the standard NCVI tests, incorporated simultaneous lower limb muscle monitoring for NMFI assessment. To accomplish such an investigation, a wide range of continuous non-invasive biomedical technologies were employed, including digital artery photoplethysmography for the extraction of cardiovascular signals, near-infrared spectroscopy for the extraction of regional tissue oxygenation in brain and muscle, and electromyography for assessment of timed muscle contractions in the lower limbs. With the novel technique described and exemplified in this paper, we were able to integrate signals from all instruments used in the assessment in a precisely synchronized fashion. We demonstrate that it is possible to visualize the interactions between all different physiological signals during the combined NCVI/NMFI assessment. Multiple counts of evidence were collected, which can capture the dynamics between skeletal muscle contractions and neurocardiovascular responses. The proposed multimodal data visualization can offer an overview of the functioning of the muscle pump during both supine rest and orthostatic recovery and can conduct comparison studies with signals from multiple participants at any given time in the assessment. This could help researchers and clinicians generate and test hypotheses based on the multimodal inspection of raw data, in long COVID and other clinical cohorts.

A Functional Human-on-a-Chip Autoimmune Disease Model of Myasthenia Gravis for Development of Therapeutics

Myasthenia gravis (MG) is a chronic and progressive neuromuscular disease where autoantibodies target essential proteins such as the nicotinic acetylcholine receptor (nAChR) at the neuromuscular junction (NMJ) causing muscle fatigue and weakness. Autoantibodies directed against nAChRs are proposed to work by three main pathological mechanisms of receptor disruption: blocking, receptor internalization, and downregulation. Current in vivo models using experimental autoimmune animal models fail to recapitulate the disease pathology and are limited in clinical translatability due to disproportionate disease severity and high animal death rates. The development of a highly sensitive antibody assay that mimics human disease pathology is desirable for clinical advancement and therapeutic development. To address this lack of relevant models, an NMJ platform derived from human iPSC differentiated motoneurons and primary skeletal muscle was used to investigate the ability of an anti-nAChR antibody to induce clinically relevant MG pathology in the serum-free, spatially organized, functionally mature NMJ platform. Treatment of the NMJ model with the anti-nAChR antibody revealed decreasing NMJ stability as measured by the number of NMJs before and after the synchrony stimulation protocol. This decrease in NMJ stability was dose-dependent over a concentration range of 0.01–20 μg/mL. Immunocytochemical (ICC) analysis was used to distinguish between pathological mechanisms of antibody-mediated receptor disruption including blocking, receptor internalization and downregulation. Antibody treatment also activated the complement cascade as indicated by complement protein 3 deposition near the nAChRs. Additionally, complement cascade activation significantly altered other readouts of NMJ function including the NMJ fidelity parameter as measured by the number of muscle contractions missed in response to increasing motoneuron stimulation frequencies. This synchrony readout mimics the clinical phenotype of neurological blocking that results in failure of muscle contractions despite motoneuron stimulations. Taken together, these data indicate the establishment of a relevant disease model of MG that mimics reduction of functional nAChRs at the NMJ, decreased NMJ stability, complement activation and blocking of neuromuscular transmission. This system is the first functional human in vitro model of MG to be used to simulate three potential disease mechanisms as well as to establish a preclinical platform for evaluation of disease modifying treatments (etiology).

Effects of dihydropyridine receptor signal on sarcoplasmic reticulum Ca2+ leakage after muscle contractions in rats

The purpose of this study is to investigate the mechanism underlying sarcoplasmic reticulum (SR) Ca2+ leakage at recovery phase after in vivo contractions. Rat gastrocnemius muscles were electrically stimulated in vivo, and then mechanically skinned fibers were prepared from the muscles excised 30 min after repeated high-intensity contractions. SR Ca2+ leakage was increased in the skinned fibers from stimulated muscles. Thereafter, SR Ca2+ leakage in skinned fibers was measured (1) under a continuously depolarized condition and (2) in the presence of nifedipine in the sealed transverse tubular system. In either of the two conditions, SR Ca2+ leakage in the rested fibers reached a level similar to that in the stimulated fibers. Furthermore, 1 mM tetracaine (Tet) treatment, but not 3 mM Mg2+ (3 Mg) treatment, lessened SR Ca2+ leakage in stimulated fibers. Depolarization-induced force in skinned fibers was more greatly decreased by Tet treatment than by 3 Mg treatment (92% reduction in Tet versus 31% reduction in 3 Mg), whereas caffeine-induced force in skinned fibers was similarly decreased by either treatment (73% reduction in Tet versus 75% reduction in 3 Mg). This difference indicates that Tet exerts a greater inhibitory effect on the dihydropyridine receptor (DHPR) signal to ryanodine receptor (RYR) than 3 Mg, although their inhibitory effects on RYR are almost similar. These results suggest that the increased Ca2+ leakage after muscle contractions is mainly caused by the orthograde signal of DHPRs to RYRs.

Changes in Medial Elbow Joint Space When Elbow Valgus Stress Is Applied at Different Limb Positions and Loads In Vivo

Background: Ulnar collateral ligament (UCL) injury is a common sports injury among overhead-throwing athletes and causes medial elbow pain and instability. UCL injury is generally diagnosed based on symptoms, physical findings, and image evaluation. To standardize the method for evaluating elbow valgus instability, more information is needed regarding changes in the medial elbow joint space (JS) in healthy elbows. Purpose/Hypothesis: The purpose of this study was to measure the JS during the application of elbow valgus stress at different elbow flexion angles and loads and to clarify the presence of defensive muscle contractions during elbow valgus stress. It was hypothesized that the JS will differ according to different limb positions and loads and that defensive contractions will occur when elbow valgus stress is >90 N. Study Design: Controlled laboratory study. Methods: Elbow joints on the nondominant side were examined in 20 healthy male university students (mean age, 21 ± 0.2 years) at 30°, 60°, and 90° of elbow flexion. To create valgus stress on the elbow, loads of 30, 60, 90, 120, and 150 N were applied with a Telos stress device and with gravity stress on the forearm. The medial JS was measured ultrasonographically during the application of elbow valgus stress. Electrodes were attached to the pronator teres muscle, and defensive muscle contractions were measured using electromyography during the application of elbow valgus stress. Repeated-measures analysis of variance and paired t tests were used to compare the JS at each elbow angle and each valgus stress load, and the Bonferroni method was used as a post hoc test. Results: At 30° of elbow flexion, the JS was significantly higher at 30 N versus 0 N and at 60 N versus 0 or 30 N ( P ≤ .018 for all). At 60° of flexion, the JS was significantly higher at 30 N versus 0 N, at 60 N versus 0 and 30 N, and at 90 N versus 0, 30, and 60 N ( P ≤ .024 for all). At 90° of elbow flexion, the JS was significantly higher at 30 N versus 0 N and at 60 N versus 0 and 30 N ( P ≤ .028 for all). Defensive muscle contraction did not occur at any elbow flexion angles at elbow valgus stress ≤60 N. Conclusion: The lack of muscular contraction at elbow valgus stress ≤60 N may reflect the function of the medial collateral ligament. Clinical Relevance: Elbow valgus stress ≤60 N allows for the evaluation of the joint opening.

EEG-Based Spectral Analysis Showing Brainwave Changes Related to Modulating Progressive Fatigue During a Prolonged Intermittent Motor Task

ABSTRACTRepeatedly performing a submaximal motor task for a prolonged period of time leads to muscle fatigue manifested by its reduced capacity to generate force or power. Fatigue resulted from voluntary muscle contractions comprises a central and peripheral component, which demands a gradually increasing effort to perform the task as time elapses. However, we still lack a complete understanding of brain contribution to the enhancement of effort to cope with progressing fatigue because of repeated submaximal muscle contractions. The knowledge of how the muscle fatigue modulates brain activities in healthy population will help rationalize why certain patients experience exacerbated fatigue while carrying out mundane tasks. The intermittent motor tasks closely resemble many activities of daily living (ADL), thus remaining physiologically relevant to study fatigue. The scope of this study is therefore to investigate the EEG-based brain activation patterns in healthy subjects performing intermittent submaximal muscle contractions until self-perceived exhaustion. Fourteen participants (median age 51.5 years; age range 26 − 72 years; 5 males) repeated elbow flexion contractions at 40% maximum voluntary contraction by following visual cues displayed on an oscilloscope screen until subjective exhaustion. Each contraction lasted ≈ 5 s with a 2-s rest between trials. The force, EEG, and surface EMG (from elbow joint muscles) data were simultaneously collected. After preprocessing, we selected a subset of trials at the beginning, middle, and end of the study session representing brain activities germane to mild, moderate, and severe fatigue conditions, respectively, to compare and contrast the changes in the EEG time-frequency (TF) characteristics across the conditions. The TF analyses were conducted both at the channel and source level. The outcome of channel- and source-level analyses reveals that the theta, alpha, and beta power spectral densities (PSDs) vary in proportion to fatigue levels in cortical motor areas. Importantly, the pairwise PSD differences between the fatigue conditions survived the statistical inferential tests with a p-value threshold of 0.05. We observed a statistically significant change in the band-specific spectral power in relation to the graded fatigue from both the steady- and post-contraction EEG data. The findings would enhance our understanding on the etiology and physiology of voluntary motor-action-related fatigue and provide pointers to counteract the perception of muscle weakness and lack of motor endurance associated with ADL. The study outcome would help evaluate how clinical conditions such as neurological disorders and cancer treatment alter neural mechanisms underlying fatigue in future studies, and develop therapeutic strategies for restoring the patients’ ability to participate in ADL by mitigating the central and muscle fatigue.