Twitch Force Research Articles

Neuromuscular recovery from severe- and extreme-intensity exercise in men and women.

Maximal voluntary contraction force (MVC), potentiated twitch force (Qpot), and voluntary activation (%VA) recover to baseline within 90s following extreme-intensity exercise. However, methodological limitations mask important recovery kinetics. We hypothesized reductions in MVC, Qpot, and %VA at task failure following extreme-intensity exercise would be less than following severe-intensity exercise, and Qpot and MVC following extreme-intensity exercise would show significant recovery within 120s but remain depressed following severe-intensity exercise. Twelve subjects (6 men) completed 2 severe-intensity (40, 50% MVC) and 2 extreme-intensity (70, 80% MVC) isometric knee-extension exercise bouts to task failure (Tlim). Neuromuscular function was measured at baseline, Tlim, and through 150s of recovery. Each intensity significantly reduced MVC and Qpot compared with baseline. MVC was greater at Tlim (p< 0.01) and at 150s of recovery (p= 0.004) following exercise at 80% MVC compared with severe-intensity exercise. Partial recovery of MVC and Qpot were detected within 150s following Tlim for each exercise intensity; Qpot recovered to baseline values within 150s of recovery following exercise at 80% MVC. No differences in %VA were detected pre- to post-exercise or across recovery for any intensity. Although further analysis showed sex-specific differences in MVC and Qpot, future studies should closely examine sex-dependent responses to extreme-intensity exercise. It is clear, however, that these data reinforce that mechanisms limiting exercise tolerance during extreme-intensity exercise recover quickly. Novelty: Severe- and extreme-intensity exercise cause independent responses in fatigue accumulation and the subsequent recovery time courses. Recovery of MVC and Qpot occurs much faster following extreme-intensity exercise in both men and women.

Neuromuscular Performance and Training Workload Over an In-Season Mesocycle in Elite Young Soccer Players.

The purpose of this study was to assess neuromuscular performance capabilities over an in-season mesocycle in early-career professional soccer players and examine the relationship with training workload. Neuromuscular performance capabilities (isometric knee extensor: peak force, rate of force development, and peak twitch force) of 12 professional soccer players were assessed weekly over a 6-week period. Training and match workload were also recorded over the same period for each player (high-intensity running distance). Changes in neuromuscular performance and workload variables were analyzed. There was evidence of fluctuations in neuromuscular performance capability over the mesocycle that reached statistical (P < .05) and practical (13.3% [peak-to-peak]) significance alongside interweek heterogeneity in training and match workload (∼17.5% [coefficient of variation], P < .05). Congruence among fluctuating patterns of intramesocycle training load and concomitant neuromuscular performance responses was noted over time for acute training load and acute:chronic workload ratio with peak force and rate of force development. Neuromuscular performance capabilities fluctuate over an in-season mesocycle and are influenced by high-intensity running workload, emphasizing the need for acute monitoring in elite soccer players.

Effects of caffeine on central and peripheral fatigue following closed- and open-loop cycling exercises.

We examined whether endurance performance and neuromuscular fatigue would be affected by caffeine ingestion during closed- and open-loop exercises. Nine cyclists performed a closed-loop (4,000-m cycling time trial) and an open-loop exercise (work rate fixed at mean power of the closed-loop trial) 60 min after ingesting caffeine (CAF, 5 mg/kg) or placebo (PLA, cellulose). Central and peripheral fatigue was quantified via pre- to post-exercise decrease in quadriceps voluntary activation and potentiated twitch force, respectively. Test sensitivity for detecting caffeine-induced improvements in exercise performance was calculated as the mean change in time divided by the error of measurement. Caffeine ingestion reduced the time of the closed-loop trial (PLA: 375.1±14.5 s vs CAF: 368.2±14.9 s, P=0.024) and increased exercise tolerance during the open-loop trial (PLA: 418.2±99.5 s vs CAF: 552.5±106.5 s, P=0.001), with similar calculated sensitivity indices (1.5, 90%CI: 0.7-2.9 vs 2.8, 90%CI: 1.9-5.1). The reduction in voluntary activation was more pronounced (P=0.019) in open- (-6.8±8.3%) than in closed-loop exercises (-1.9±4.4%), but there was no difference between open- and closed-loop exercises for the potentiated twitch force reduction (-25.6±12.8 vs -26.6±12.0%, P>0.05). Caffeine had no effect on central and peripheral fatigue development in either mode of exercise. In conclusion, caffeine improved endurance performance in both modes of exercise without influence on post-exercise central and peripheral fatigue, with the open-loop exercise imposing a greater challenge to central fatigue tolerance.

Airflow restriction mask induces greater central fatigue after a non-exhaustive high-intensity interval exercise.

The airflow restriction mask (ARM) is a practical and inexpensive device for respiratory muscle training. Wearing an ARM has recently been combined with high-intensity interval exercise (HIIE), but its effect on neuromuscular fatigue is unknown. The present study investigated the effects of ARM wearing on neuromuscular fatigue after an HIIE session. Fourteen healthy men performed two HIIE sessions (4×4min at 90% HRmax , 3min recovery at 70% HRmax ) with or without an ARM. Neuromuscular fatigue was quantified via pre- to post-HIIE changes in maximal voluntary contraction (MVC), voluntary activation (VA, central fatigue), and potentialized evoked twitch force at 100, 10, and 1Hz (peripheral fatigue). Blood pH and lactate were measured before and after the HIIE session, while HR, SpO2 , dyspnea, physical sensation of effort (P-RPE), and Task Effort and Awareness (TEA) were recorded every bout. The exercise-induced decrease in MVC was higher (p<0.05) in the ARM (-28±12%) than in the Control condition (-20±11%). The VA decreased (p<0.05) in the ARM (-11±11%) but not in the control condition (-4±5%, p>0.05). Pre- to post-HIIE declines in evoked twitch at 100, 10, and 1Hz were similar (p>0.05) between ARM and control conditions (ARM: -18±10, -43±11 and -38±12%; Control: -18±14, -43±12 and -37±17%). When compared with the control, the HIIE bout wearing ARM was marked by higher heart rate, plasma lactate concentration, dyspnea, P-RPE and TEA, as well as lower SpO2 and blood pH. In conclusion, ARM increases perceptual and physiological stress during a HIIE, which may lead to a greater post-exercise central fatigue.

Reading Frame Repair of TTN Truncation Variants Restores Titin Quantity and Functions.

Titin truncation variants (TTNtvs) are the most common inheritable risk factor for dilated cardiomyopathy (DCM), a disease with high morbidity and mortality. The pathogenicity of TTNtvs has been associated with structural localization as A-band variants overlapping myosin heavy chain-binding domains are more pathogenic than I-band variants by incompletely understood mechanisms. Demonstrating why A-band variants are highly pathogenic for DCM could reveal new insights into DCM pathogenesis, titin (TTN) functions, and therapeutic targets. We constructed human cardiomyocyte models harboring DCM-associated TTNtvs within A-band and I-band structural domains using induced pluripotent stem cell and CRISPR technologies. We characterized normal TTN isoforms and variant-specific truncation peptides by their expression levels and cardiomyocyte localization using TTN protein gel electrophoresis and immunofluorescence, respectively. Using CRISPR to ablate A-band variant-specific truncation peptides through introduction of a proximal I-band TTNtv, we studied genetic mechanisms in single cardiomyocyte and 3-dimensional, biomimetic cardiac microtissue functional assays. Last, we engineered a full-length TTN protein reporter assay and used next-generation sequencing assays to develop a CRISPR therapeutic for somatic cell genome editing TTNtvs. An A-band TTNtv dose-dependently impaired cardiac microtissue twitch force, reduced full-length TTN levels, and produced abundant TTN truncation peptides. TTN truncation peptides integrated into nascent myofibril-like structures and impaired myofibrillogenesis. CRISPR ablation of TTN truncation peptides using a proximal I-band TTNtv partially restored cardiac microtissue twitch force deficits. Cardiomyocyte genome editing using SpCas9 and a TTNtv-specific guide RNA restored the TTN protein reading frame, which increased full-length TTN protein levels, reduced TTN truncation peptides, and increased sarcomere function in cardiac microtissue assays. An A-band TTNtv diminished sarcomere function greater than an I-band TTNtv in proportion to estimated DCM pathogenicity. Although both TTNtvs resulted in full-length TTN haploinsufficiency, only the A-band TTNtv produced TTN truncation peptides that impaired myofibrillogenesis and sarcomere function. CRISPR-mediated reading frame repair of the A-band TTNtv restored functional deficits, and could be adapted as a one-and-done genome editing strategy to target ≈30% of DCM-associated TTNtvs.

Extensor carpi ulnaris muscle shows unexpected slow-to-fast fiber-type switch in Duchenne muscular dystrophy dogs.

ABSTRACTAged dystrophin-null canines are excellent models for studying experimental therapies for Duchenne muscular dystrophy, a lethal muscle disease caused by dystrophin deficiency. To establish the baseline, we studied the extensor carpi ulnaris (ECU) muscle in 15 terminal age (3-year-old) male affected dogs and 15 age/sex-matched normal dogs. Affected dogs showed histological and anatomical hallmarks of dystrophy, including muscle inflammation and fibrosis, myofiber size variation and centralized myonuclei, as well as a significant reduction of muscle weight, muscle-to-body weight ratio and muscle cross-sectional area. To rigorously characterize the contractile properties of the ECU muscle, we developed a novel in situ assay. Twitch and tetanic force, contraction and relaxation rate, and resistance to eccentric contraction-induced force loss were significantly decreased in affected dogs. Intriguingly, the time-to-peak tension and half-relaxation time were significantly shortened in affected dogs. Contractile kinetics predicted an unforeseen slow-to-fast myofiber-type switch, which we confirmed at the protein and transcript level. Our study establishes a foundation for studying long-term and late-stage therapeutic interventions in dystrophic canines. The unexpected myofiber-type switch highlights the complexity of muscle remodeling in dystrophic large mammals. This article has an associated First Person interview with the first author of the paper.

In vivo potentiation of post-tetanic twitch across age and sex

Twitch force potentiation of fast-twitch skeletal muscle is produced by repetitive stimulation that can be achieved from either (1) the staircase effect (continual low frequency stimulation) or (2) post-tetanic potentiation (a 1-2 s high-frequency tetanic stimulation). Previous studies examining twitch force potentiation have been conducted in vitro and shown that it is related to phosphorylation of myosin regulatory light chain (pRLC). We previously found, in vitro, reduced potentiation of twitch force and decreased pRLC in ovariectomized (Ovx, estrogen-deficient) compared with sham-operated (estrogen-replete) mice. Thus, we questioned whether this phenomenon occurred in vivo and whether age and sex would affect the potentiation of twitch force. Using an in vivo post-tetanic potentiation method (one twitch contraction followed by a tetanic contraction-100 Hz for 1,000 ms with 0.01 ms pulses, and two post-tetanic twitch contractions), we investigated twitch torque potentiation in C57BL/6 young and old, male and female mice. There were significant main effects of sex (P < 0.001) and age (P < 0.001) on body mass and significant main effects of sex (P < 0.001) on tibialis anterior and extensor digitorum longus muscle masses, with males and aged being relatively greater. Analysis of twitch torque using a three-way ANOVA across time, age, and sex showed a significant main effect of time (pre < post; P < 0.001), time × age (P = 0.038), and time × sex (P = 0.028), indicating potentiation occurred in young and old, males and females. Analysis of twitch torque potentiation (percent increase) using a two-way ANOVA revealed a significant main effect of age (young = 45.16 ± 2.04 versus old = 27.88 ± 9.96; P < 0.001) with no effect of sex (P = 0.215). In summary, enhanced generation of twitch force of skeletal muscle using a post-tetanic potentiation method does occur in vivo and is affected by age but not sex, as there is greater twitch torque potentiation in young than old mice.

Decoding muscle force from individual motor unit activities using a twitch force model and hybrid neural networks

A deep learning method involving hybrid neural networks incorporated with a physiological twitch force model is presented for decoding muscle force from individual motor unit (MU) activities. A series of MU action potential (MUAP) trains were obtained using a progressive FastICA peel-off algorithm through high-density surface electromyogram (HD-sEMG) decomposition. The twitch force model was adopted to transform MUAP trains into twitch force sequences. The hybrid convolutional neural networks (CNNs) consisting of a spatial attention model (SA-CNN) and a bidirectional gated recurrent unit (Bi-GRU) were fed by the twitch force sequences to predict the muscle force. The performance of the proposed method was evaluated in predicting the thumb abduction force using both simulated data and experimental HD-sEMG recordings from abductor pollicis brevis muscles of ten subjects. The simulation approach assisted our method design in confirming a simplified relationship between MUAP amplitude and force contribution of each MU. When processing experimental data, the root mean square deviation (RMSD) values between the predicted and the actual forces were 6.57% ± 1.26% and 7.18% ± 0.96% under the user-specific testing scheme and the user-independent testing scheme, respectively. The proposed method outperformed other common sEMG amplitude-based or MU-based methods with significantly decreased RMSD values (p < 0.001). These findings indicate the advance of mining spatial information from individual MU activities using deep neural networks in decoding the muscle force. This study provides a useful tool for muscle force estimation, with wide applications in biomechanics and sports rehabilitation.

Angiotensin 1-7 prevents the excessive force loss resulting from 14- and 28-day denervation in mouse EDL and soleus muscle.

Denervation leads to muscle atrophy, which is described as muscle mass and force loss, the latter exceeding expectation from mass loss. The objective of this study was to determine the efficiency of angiotensin (Ang) 1-7 at reducing muscle atrophy in mouse extensor digitorum longus (EDL) and soleus following 14- and 28-d denervation periods. Some denervated mice were treated with Ang 1-7 or diminazene aceturate (DIZE), an ACE2 activator, to increase Ang 1-7 levels. Ang 1-7/DIZE treatment had little effect on muscle mass loss and fiber cross-sectional area reduction. Ang 1-7 and DIZE fully prevented the loss of tetanic force normalized to cross-sectional area and accentuated the increase in twitch force in denervated muscle. However, they did not prevent the shift of the force-frequency relationship toward lower stimulation frequencies. The Ang 1-7/DIZE effects on twitch and tetanic force were completely blocked by A779, a MasR antagonist, and were not observed in MasR-/- muscles. Ang 1-7 reduced the extent of membrane depolarization, fully prevented the loss of membrane excitability, and maintained the action potential overshoot in denervated muscles. Ang 1-7 had no effect on the changes in α-actin, myosin, or MuRF-1, atrogin-1 protein content or the content of total or phosphorylated Akt, S6, and 4EPB. This is the first study that provides evidence that Ang 1-7 maintains normal muscle function in terms of maximum force and membrane excitability during 14- and 28-d periods after denervation.

Sarcolemmal Excitability, M-Wave Changes, and Conduction Velocity During a Sustained Low-Force Contraction.

This study was undertaken to investigate whether sarcolemmal excitability is impaired during a sustained low-force contraction [10% maximal voluntary contraction (MVC)] by assessing muscle conduction velocity and also by analyzing separately the first and second phases of the muscle compound action potential (M wave). Twenty-one participants sustained an isometric knee extension of 10% MVC for 3min. M waves were evoked by supramaximal single shocks to the femoral nerve given at 10-s intervals. The amplitude, duration, and area of the first and second M-wave phases were computed. Muscle fiber conduction velocity, voluntary surface electromyographic (EMG), perceived effort, MVC force, peak twitch force, and temperature were also recorded. The main findings were: (1) During the sustained contraction, conduction velocity remained unchanged. (2) The amplitude of the M-wave first phase decreased for the first ~30s (−7%, p<0.05) and stabilized thereafter, whereas the second phase amplitude increased for the initial ~30s (+7%, p<0.05), before stabilizing. (3) Both duration and area decreased steeply during the first ~30s, and then more gradually for the rest of the contraction. (4) During the sustained contraction, perceived effort increased fivefold, whereas knee extension EMG increased by ~10%. (5) Maximal voluntary force and peak twitch force decreased (respectively, −9% and −10%, p<0.05) after the low-force contraction. Collectively, the present results indicate that sarcolemmal excitability is well preserved during a sustained 10% MVC task. A depression of the M-wave first phase during a low-force contraction can occur even in the absence of changes in membrane excitability. The development of fatigue during a low-force contraction can occur without alteration of membrane excitability.

Development and reliability of a new system for bedside evaluation of non-volitional knee extension force

PurposeNeuromuscular electrical stimulation (NMES) is a widely-used technique for diagnostic and therapeutic purposes. Here we developed and tested the reliability of a new NMES-dynamometer system for bedside evaluation of knee extensor muscle function. Materials and methodsThirty-two healthy participants (16 men, 16 women; 27±5 years) completed two testing sessions, 7 days apart. On day 1, a single experienced rater, who repeated the evaluation on day 2 with two other raters, completed a standardized testing procedure. Participants were placed supine, with knees flexed and legs connected to the dynamometer. Maximal voluntary knee extensor isometric force (MVF) and supramaximal twitch force (TwF) were obtained. ResultsHigh intra-rater intraclass correlation coefficients were observed for both MVF (0.91) and TwF (0.94). MVF and TwF standard error of measurements (8.2%, 5.9%) and minimal detectable changes (16%, 11.6%) were low compared to mean values. High intraclass correlation coefficients were also observed for inter-rater comparisons of MVF (0.89) and TwF (0.86). Standard errors of measurements (MVF: 8.7%, TwF: 5.5%) and minimal detectable changes (MVF: 17.2%, TwF: 10.8%) were similar to intra-rater comparisons. ConclusionThe good reliability of the novel NMES-dynamometer system suggests it as an appropriate tool for the bedside evaluation of knee extensor muscle function.

Potassium-induced potentiation of subtetanic force in rat skeletal muscles: influences of β2-activation, lactic acid, and temperature.

Moderate elevations of extracellular K+ concentration ([K+]o) occur during exercise and have been shown to potentiate force during contractions elicited with subtetanic frequencies. Here, we investigated whether lactic acid (reduced chloride conductance), β2-adrenoceptor activation, and increased temperature would influence the potentiating effect of potassium in slow- and fast-twitch muscles. Isometric contractions were elicited by electrical stimulation at various frequencies in isolated rat soleus and extensor digitorum longus (EDL) muscles incubated at normal (4 mM) or elevated K+, in combination with salbutamol (5 μM), lactic acid (18.1 mM), 9-anthracene-carboxylic acid (9-AC; 25 μM), or increased temperature (30-35°C). Elevating [K+]o from 4 mM to 7 mM (soleus) and 10 mM (EDL) potentiated isometric twitch and subtetanic force while slightly reducing tetanic force. In EDL, salbutamol further augmented twitch force (+27 ± 3%, P < 0.001) and subtetanic force (+22 ± 4%, P < 0.001). In contrast, salbutamol reduced subtetanic force (-28 ± 6%, P < 0.001) in soleus muscles. Lactic acid and 9-AC had no significant effects on isometric force of muscles already exposed to moderate elevations of [K+]o. The potentiating effect of elevated [K+]o was still well maintained at 35°C. Addition of salbutamol exerts a further force-potentiating effect in fast-twitch but not in slow-twitch muscles already potentiated by moderately elevated [K+]o, whereas lactic acid, 9-AC, or increased temperature does not exert any further augmentation. However, the potentiating effect of elevated [K+]o was still maintained in the presence of these, thus emphasizing the positive influence of moderately elevated [K+]o for contractile performance during exercise.

Evidence that Neuromuscular Fatigue Is not a Dogma in Patients with Parkinson's Disease.

Given the increased level of fatigue frequently reported by patients with Parkinson's disease (PD), this study investigated the interaction between central and peripheral components of neuromuscular fatigue (NF) in this population compared with healthy peers. Changes in maximal voluntary activation (ΔVA, central fatigue) and potentiated twitch force (ΔQtw,pot, peripheral fatigue) pre-post exercise were determined via the interpolated twitch technique in 10 patients with PD and 10 healthy controls (CTRL) matched for age, sex, and physical activity. Pulmonary gas exchange, femoral blood flow, and quadriceps EMG were measured during a fatiguing exercise (85% of peak power output [PPO]). For a specific comparison, on another day, CTRL repeat the fatiguing test matching the time to failure (TTF) and PPO of PD. At 85% of PPO (PD, 21 ± 7 W; CTRL, 37 ± 22 W), both groups have similar TTF (~5.9 min), pulmonary gas exchange, femoral blood flow, and EMG. After this exercise, the maximal voluntary contraction (MVC) force and Qtwpot decreased equally in both groups (-16%, P = 0.483; -43%, P = 0.932), whereas VA decreased in PD compared with CTRL (-3.8% vs -1.1%, P = 0.040). At the same PPO and TTF of PD (21 W; 5.4 min), CTRL showed a constant drop in MVC, and Qtwpot (-14%, P = 0.854; -39%, P = 0.540), instead VA decreased more in PD than in CTRL (-3.8% vs -0.7%, P = 0.028). In PD, central NF seems exacerbated by the fatiguing task which, however, does not alter peripheral fatigue. This, besides the TTF like CTRL, suggests that physical activity may limit NF and counterbalance PD-induced degeneration through peripheral adaptations.

The Safety Factor for Neuromuscular Transmission: Effects of Dimethylsulphoxide, Cannabinoids and Synaptic Homeostasis.

BackgroundIn myasthenia gravis, impaired postsynaptic sensitivity to acetylcholine results in failure of neuromuscular transmission and fatiguing muscle weakness.ObjectiveDevelop an ex vivo muscle contraction assay to test cannabinoids and other substances that might act on the myasthenic neuromuscular junction to restore control of the muscle.MethodsTubocurarine was added to an ex vivo, mouse phrenic nerve-hemidiaphragm muscle preparation to reduce acetylcholine sensitivity. This produced a myasthenia-like decrement in twitch force during a train of 10 nerve impulses (3 / sec). Endplate potential (EPP) recordings were used to confirm and extend the findings.ResultsSurprisingly, addition to the bath of dimethylsulphoxide (DMSO), at concentrations as low as 0.1%(v/v), partially reversed the decrement in nerve-evoked force. Intracellular electrophysiology, conducted in the presence of tubocurarine, showed that DMSO increased the amplitudes of both the spontaneous miniature EPP (MEPP) and the (nerve-evoked) EPP. In the absence of tubocurarine (synaptic potentials at physiological levels), an adaptive fall in quantal content negated the DMSO-induced rise in EPP amplitude. The effects of cannabinoid receptor agonists (solubilized with DMSO) in the contraction assay do not support their further exploration as useful therapeutic agents for myasthenia gravis. CP 55,940 (a dual agonist for cannabinoid receptor types 1 and 2) reversed the beneficial effects of DMSO.Conclusions:We demonstrate a powerful effect of DMSO upon quantal amplitude that might mislead pharmacological studies of synaptic function wherever DMSO is used as a drug vehicle. Our results also show that compounds targeting impaired neuromuscular transmission should be tested under myasthenic-like conditions, so as to avoid confounding effects of synaptic homeostasis.

Recovery From Eccentric Squat Exercise in Resistance-Trained Young and Master Athletes With Similar Maximum Strength: Combining Cold Water Immersion and Compression.

The aim of this study was to investigate whether recovery from eccentric squat exercise varies depending on age and to assess whether the use of a mixed-method recovery (MMR) consisting of cold water immersion and compression tights benefits recovery. Sixteen healthy and resistance-trained young (age, 22.1±2.1years; N=8) and master male athletes (age, 52.4±3.5years; N=8), who had a similar half squat 1-repetition maximum relative to body weight, completed two identical squat exercise training sessions, separated by a 2-week washout period. Training sessions were followed by either MMR or passive recovery (PR). Internal training loads [heart rate and blood lactate concentration (BLa)] were recorded during and after squat sessions. Furthermore, maximal voluntary isometric contraction (MVIC) force, countermovement jump (CMJ) height, resting twitch force of the knee extensors, serum concentration of creatine kinase (CK), muscle soreness (MS), and perceived physical performance capability (PPC) were determined before and after training as well as after 24, 48, and 72h of recovery. A three-way mixed ANOVA revealed a significant time effect of the squat protocol on markers of fatigue and recovery (p<0.05; decreased MVIC, CMJ, twitch force, and PPC; increased CK and MS). Age-related differences were found for BLa, MS, and PPC (higher post-exercise fatigue in younger athletes). A significant two-way interaction between recovery strategy and time of measurement was found for MS and PPC (p<0.05; faster recovery after MMR). In three participants (two young and one master athlete), the individual results revealed a consistently positive response to MMR. In conclusion, master athletes neither reach higher fatigue levels nor recover more slowly than the younger athletes. Furthermore, the results indicate that MMR after resistance exercise does not contribute to a faster recovery of physical performance, neuromuscular function, or muscle damage, but promotes recovery of perceptual measures regardless of age.

Abstract P491: Myosin-7 E848G Mutation Induces P53-associated Cell Death That Leads To Impaired Tissue Contractility In Patient-derived Induced Pluripotent Stem Cell Model Of Hypertrophic Cardiomyopathy

Introduction: Familial hypertrophic cardiomyopathy (HCM), affecting 1 in 500 adults, is characterized by idiopathic thickening of the heart and occasional impaired systolic function. Mechanisms through which cardiac sarcomeric mutations manifest in HCM are poorly understood. Hypothesis: We previously identified a novel MYH7 E848G mutation associated with HCM. We hypothesize E848G induces cell death that results in impaired tissue contractility in a dose-dependent manner. Methods: We created MYH7 expressing CMs with WT/WT, E848G/WT, or E848G/E848G alleles by CRISPR/Cas9 gene-editing patient-specific induced pluripotent stem cells (hiPSCs). hiPSC-derived cardiomyocytes were metabolically purified and cocultured with stromal cells on PDMS posts to create 3D engineered heart tissues (EHTs) or cultured as a monolayer. Results: Day 65 monolayer E848G/E848G CMs had 48.5% effective cell number relative to WT/WT. p53 (2.80 ± .11-fold), p21 (7.24 ± .18-fold), and BAX (1.64 ± 0.14-fold) mRNA transcripts were upregulated in day 60 monolayer E848G/E848G relative to WT/WT. E848G/E848G EHTs (n = 12) exhibited lower maximum active twitch force (104.6 ± 18.2 μN) and smaller 2D projected area (5.31 ± 0.22 mm 2 ) at day 14 relative to WT/WT (n = 15; 238.0 ± 20.4 μN; 6.87 ± 0.26 mm 2 ). E848G/WT EHTs (n = 7) had intermediate twitch force (168.7 ± 12.2 μN) and 2D area (6.18 ± 0.36 mm 2 ). Conclusion: These results suggest the MYH7 E848G mutation induces p53-associated cell death that leads to reduced tissue contractility. Ongoing studies will elucidate the molecular mechanism through which E848G activates cell death pathways. Figure: Representative EHTs. L-R: WT/WT, E848G/WT, E848G/E848G.

Abstract P459: Mavacamten And Danicamtiv Reverse Respective Contractile Abnormalities In Engineered Heart Tissue Models Of Hypertrophic And Dilated Cardiomyopathy

Missense mutations in alpha-tropomyosin (TPM1) can lead to development of hypertrophic (HCM) or dilated cardiomyopathy (DCM). HCM mutation E62Q and DCM mutation E54K have previously been studied extensively in experimental systems ranging from in vitro biochemical assays to animal models, although some conflicting results have been found. We undertook a detailed multi-scale assessment of these mutants that included atomistic simulations, regulated in vitro motility (IVM) assays, and finally physiologically relevant human engineered heart tissues. In IVM assays, E62Q previously has shown increased Calcium sensitivity. New molecular dynamics data shows mutation-induced changes to tropomyosin dynamics and interactions with actin and troponin. Human engineered heart tissues (EHT) were generated by seeding iPSC-derived cardiomyocytes engineered using CRISPR/CAS9 to express either E62Q or E54K cardiomyopathy mutations. After two weeks in culture, E62Q EHTs showed a drastically hypercontractile twitch force and significantly increased stiffness while displaying little difference in twitch kinetics compared to wild-type isogenic control EHTs. On the other hand, E54K EHTs displayed hypocontractile isometric twitch force with faster kinetics, impaired length-dependent activation and lowered stiffness. Given these contractile abnormalities, we hypothesized that small molecule myosin modulators to appropriately activate or inhibit myosin activity would restore E54K or E62Q EHTs to normal behavior. Accordingly, E62Q EHTs were treated with 0.5μM mavacamten (to remedy hypercontractility) and E54K EHTs with 0.5 μM danicamtiv (to remedy hypocontractility) for 4 days, followed by a 1 day washout period. Upon contractility testing, it was observed that the drugs were able to reverse contractile phenotypes observed in mutant EHTs and restore contractile properties to levels resembling those of the untreated wild type group. The computational, IVM and EHT studies provide clear evidence in support of the hyper- vs. hypo-contractility paradigm as a common axis that distinguishes HCM and DCM TPM1 mutations. Myosin modulators that directly compensate for underlying myofilament aberrations show promising efficacy in human in vitro systems.

QS6: Collagen-glycosaminoglycans Scaffold Can Improve Muscle Function Recovery After Volumetric Muscle Loss Injury in Rat Model

Propose:Skeletal muscle regenerative capacity fails in restoring muscle function following major injuries in which significant skeletal muscle is lost or damaged. As such, volumetric muscle loss (VML), ultimately results in permanent disability. Therapeutic options are limited in VML and currently no standard of care exists. Biodegradable scaffolds hold great potentials in the regeneration of lost muscle tissue by replacing the lost structural framework of the defect. In this study we aim to evaluate the effects of a porous collagen-glycosaminoglycans scaffold (CGS) on muscle function recovery following VML injury in a rat model.Methods:Fifteen male 12-week old Sprague-Dawley rats were randomly divided into three groups of 5 rats in each: 1) Sham group, 2) VML Untreated (control) group, and 3) VML+CGS group. A standard model of VML injury was performed to bilateral Tibialis Anterior (TA) muscles in VML Untreated and VML+CGS groups. The muscle defect in VML+CGS group was filled with 3 sheets of CGS cuts while the muscle defects in VML control group left untreated. Animals in Sham group underwent the same surgical procedure (skin and fascia opening and closure) but without any muscle injury. Pick isometric twitch and tetanus forces of foot dorsiflexion were measured in vivo prior, immediately after, and four weeks post injury using a dual mode muscle lever system. In situ TA muscle twitch and tetanic contraction strengths were also determined six weeks after injury.Results:Prior, and immediately after injury no statistically significant differences was seen between VML Untreated and VML+CGS groups in terms of the means of twitch and tetanus foot isometric dorsiflexion forces. Four weeks after injury, TA of VML+ CGS group showed significant functional recovery as compared with VML Untreated group in twitch (76 ± 22.7% vs 51.3 ± 22.3%, p<0.01) and tetanus (77.4 ± 22.1% vs 59.8 ± 22.4%, p<0.05) foot dorsiflexion forces. In situ muscle strength measurements of TA at six weeks post injury, also showed significantly higher twitch (35.2 ± 12.5 mN/mm2 vs 23.7 ± 6.9 mN/mm2, p<0.05) and tetanus (115.0 ± 35.3 mN/mm2 vs 87.9 ± 20 mN/mm2, p<0.05) forces in TA of VML+CGS group when compared with the VML Untreated group. On histology of the TA performed six weeks following initial injury, placement of CGS decreased fibrosis across the injury, as defined as percentage of positive area in trichrome staining compared with VML Untreated group (1.3 ± 0.1% vs. 7.1 ± 1.3%, p<0.01). In addition, immunofluorescence tissue staining confirmed positive myosin heavy chain staining within the scaffold, indicating the formation of nascent myofibers within the area of injury.Conclusions:Application of CGS to the muscle defect following VML injury can improve muscle function recovery, limit fibrosis, and promote muscle regeneration in rodent model. These findings can foster future research in larger animals and human subjects.*p<0.05, **p<0.01. Error bars: Standard Deviations.

QS7: Physiologic Signaling of the Muscle Cuff Regenerative Peripheral Nerve Interface (MC-RPNI) During Volitional Behavior

Purpose:Exoskeletons have become a promising device to restore extremity function to those with limb weakness. However, these devices have not become widely adopted due to the inadequacy of current nerve interfacing methods. The Muscle Cuff Regenerative Peripheral Nerve Interface (MC-RPNI) was developed as a potential solution to this problem. Consisting of a segment of autologous free muscle secured around an intact nerve, the MC-RPNI is able to regenerate and reinnervate, amplifying its contained nerve’s signals through generation of EMG signals. These amplified, high-fidelity signals can then be used to intuitively and accurately control exoskeletons. The purpose of this study was to characterize MC-RPNI physiologic signaling during volitional behavior and determine long-term effects on the associated nerve.Methods:Eighteen rats were randomly assigned to one of three groups: (1) sham surgery/control; (2) nerve transection; and (3) MC-RPNI. MC-RPNIs were surgically fabricated by wrapping isogenic donor muscle graft circumferentially around the common peroneal (CP) nerve. At six months, CP nerve cuff electrodes were implanted in Groups 1 and 2,and patch electrodes were placed on all MC-RPNIs. Gait analysis and electrophysiological evaluations were performed the following day. Rats were trained to walk on a treadmill, and electrode recordings were obtained and correlated with gait videocapture. All rats had their proximal CP nerve stimulated, with efferent signals obtained at (1) downstream nerve (CSNAP), (2) MC-RPNI (CMAP), and (3) downstream-innervated extensor digitorum longus (EDL) muscle (CMAP). EDL muscle force testing was also performed following stimulation of the CP nerve.Results:All MC-RPNIs remained viable and demonstrated appropriate regeneration, revascularization, and reinnervation on histology. The MC-RPNI was found to generate large-amplitude CMAPs (2.77+/-0.926mV), amplifying its associated nerve’s signal (50.5+/-8.18µV) over 50-fold on average. The MC-RPNI was not found to affect muscle function when evaluating downstream-innervated EDL CMAPs (control: 13.4+/-2.33mV vs MC-RPNI: 14.1+/-1.44mV) or maximal twitch force (control: 562+/-70.8mN vs MC-RPNI: 653+/-34.6mN). On gait analysis, recordings from the MC-RPNI correlated with the toe-off phase of gait; for the control groups, nerve signaling could not be differentiated from background noise. When comparing MC-RPNI to control animal gait, no significant differences were noted on qualitative or joint-angle analysis.Conclusion:The MC-RPNI has the ability to chronically amplify physiologic nerve signals from intact peripheral nerves by several magnitudes while avoiding functional impairment of downstream-innervated muscle. This amplification has the capability to facilitate high accuracy detection of motor intent in order to intuitively and reliably control advanced exoskeleton devices.

Characterization of LRP4/Agrin Antibodies From a Patient With Myasthenia Gravis.

To determine whether human anti-LRP4/agrin antibodies are pathogenic in mice and to investigate underpinning pathogenic mechanisms. Immunoglobulin (Ig) was purified from a patient with myasthenia gravis (MG) with anti-LRP4/agrin antibodies and transferred to mice. Mice were characterized for body weight, muscle strength, twitch and tetanic force, neuromuscular junction (NMJ) functions including compound muscle action potential (CMAP) and endplate potentials, and NMJ structure. Effects of the antibodies on agrin-elicited muscle-specific tyrosine kinase (MuSK) activation and AChR clustering were studied and the epitopes of these antibodies were identified. Patient Ig-injected mice had MG symptoms, including weight loss and muscle weakness. Decreased CMAPs, reduced twitch and tetanus force, compromised neuromuscular transmission, and NMJ fragmentation and distortion were detected in patient Ig-injected mice. Patient Ig inhibited agrin-elicited MuSK activation and AChR clustering. The patient Ig recognized the β3 domain of LRP4 and the C-terminus of agrin and reduced agrin-enhanced LRP4-MuSK interaction. Anti-LRP4/agrin antibodies in the patient with MG is pathogenic. It impairs the NMJ by interrupting agrin-dependent LRP4-MuSK interaction.