Experimental Physiology

Cellular viscoelastic modulus in skeletal muscle tissue responds dynamically to chronic stressors, such as age and exercise. Passive tissue mechanics may also be sensitive to acute stimuli such as mechanical loading and/or activation-induced muscle fatigue. These insights are largely derived from preclinical studies of age and acute muscle activation. Therefore, we sought to understand the relative responsiveness of muscle cellular passive mechanics to chronic (resistance training) and acute (muscle fatigue) stressors in healthy young males and females categorized as "resistance trained" or "untrained". We measured passive mechanics to test the hypothesis that Youngs Modulus and stress would be greater in fibers from trained versus untrained participants and both would be reduced following fatigue. We further assessed the translation of these findings to composite tissue in a sub-set of volunteers where muscle tissue bundles, containing both fibers and extracellular matrix, were analyzed in addition to single fibers. We report a main effect of training such that cellular passive mechanical measures were increased in single fibers from trained versus untrained participants. We likewise report reductions in passive mechanical measures following fatiguing exercise. Surprisingly, both training and acute fatigue only impacted muscle fiber passive measures in males, whereas females showed a more variable response across conditions. Last, we provide preliminary evidence supporting the translation of per-individual cellular differences to the tissue level. Together, these data suggest males respond more dynamically to acute and chronic stressors of muscle tissue mechanics, potentially linking cellular response and sex-dependent differences in musculotendinous injury risk. New and noteworthyWe report that passive stress and modulus in single muscle fibers was higher in resistance trained healthy adults and fatiguing exercise reduced passive stress and modulus. In each case, dynamic responsiveness of muscle fibers to chronic and acute stressors was observed consistently in males, whereas responses in females varied considerably. We provide further evidence that cellular mechanisms may contribute to multicellular muscle tissue samples, suggesting these findings have relevance to in vivo tissue mechanics.

Plantar tissue adaptation during activity is thought to contribute to diabetic foot ulceration (DFU), yet most existing studies only measure compressive quasi-static properties. This pilot study developed an ultrasound-loadcell measurement tool, PlantarSense, and used an infrared thermometer to measure dynamic compressive and shear energy dissipation ratio (EDR) and temperature of plantar-tissue at the first metatarsal head (1stMTH) and calcaneus in people living with and without diabetes at baseline, post-walk, and post-recovery. People living with diabetes showed significantly greater post-walk temperature increases (11.0 % vs 6.9% in controls at calcaneus, p=0.03) and less complete thermal recovery than controls. Baseline compressive EDR at the 1stMTH was significantly higher in people living with diabetes (67.8% vs 56.0% in controls, p=0.04). EDR modulation was greater from shear loading (21.5%) than compression (5.4%) and post-walk induced reductions in EDR were present in all participants, but people living with diabetes showed a 20% lower recovery than controls. Impaired thermoregulation and tissue adaptation in people living with diabetes was demonstrated by plantar temperature and EDR differences in post-walk and post-recovery. Future work is needed to test more participants with a greater range of diabetes progression to quantify statistically significant plantar tissue differences to inform DFU risk management.

We investigated the effects of chemically-induced ovarian failure on single muscle fibre contractility of the soleus and extensor digitorum longus (EDL) muscles throughout ovarian failure, thereby mimicking the menopausal transition into late-stage menopause: [(D60;peri-menopause), (D120;onset of menopause), (D134;early-onset menopause), (D176;late-stage menopause)]. We used 4-vinylcyclohexene diepoxide (VCD) to induce ovarian failure in sexually-mature female mice. At D120 and D176, mice with VCD-induced ovarian failure produced higher force as compared with controls (p<0.05). On D134, however, VCD had lower force production compared with controls (p<0.05). The cross-sectional area of the soleus fibres from the VCD group was larger at D120 compared with controls (p<0.05), but not at any other time point (p>0.05). As well, at D120-D176, the proportion of Type II fibres relative to Type I increased for the soleus, but not the EDL. No differences in rate of force redevelopment (Ktr) was observed for the soleus (p>0.05), while calcium sensitivity increased by late-stage menopause (p<0.05). There were no differences in force, cross-sectional area, stiffness, Ktr, or calcium sensitivity between groups for the EDL (p>0.05). Muscle contractility across the peri-menopausal transition into late-stage menopause is both muscle and phase-dependent, emphasizing the complexity of changing hormones throughout the lifespan on muscle contractile function.

IntroductionHealthy, resting skeletal muscle primarily oxidizes lipid, but insulin resistant muscle oxidizes carbohydrate and shows metabolic inflexibility during hyperinsulinemia. It is unclear whether fuel selection and metabolic flexibility are dependent on insulin sensitivity in skeletal muscle performing mild exercise. Research Design and MethodsSedentary volunteers underwent a cycle exercise protocol using stepwise increments in power output (15, 30, and 45 watts) and indirect calorimetry to estimate fuel oxidation in working muscle. Euglycemic clamps, indirect calorimetry and muscle biopsies were used to measure insulin sensitivity and acetylation and content of Adenine Nucleotide Translocase 1 (ANT1), which might be involved in fuel selection via acetylation of lysine 23, which was quantified using mass spectrometry. ResultsMild exercise produced predicted rates of oxygen consumption (11-12 ml O2/min), with low and stable blood lactate, allowing use of indirect calorimetry to calculate a respiratory exchange ratio in working muscle (RERm). ANT1 acetylation varied from 0.6 to 21% (10.3 {+/-} 1.2%). Exercising muscle mainly oxidized carbohydrate (45 {+/-} 9, 62 {+/-} 6, and 70 {+/-} 5% of total at 15, 30, and 45watts). Multiple linear regression showed that RERm rose with increasing power output (P < 0.001) and was lower with greater protein content of ANT1 (P < 0.001). Insulin-stimulated glucose disposal, ANT acetylation, and VO2peak were not predictors of RERm. ConclusionsMildly exercising muscle in sedentary people prefers to oxidize carbohydrate independent of insulin sensitivity but depending on ANT1 protein content. The ability to oxidize lipid may be regulated by higher ANT1 content due to either higher mitochondrial abundance or greater ANT content per mitochondrial mass.

Skeletal muscle is a plastic tissue that adapts to exercise through fusion of muscle stem cells (MuSCs) with myofibers, a physiological process referred to as myonuclear accretion. However, it is still unclear whether myonuclear accretion is driven by increased mechanical loading per se, or occurs, at least in part, in response to exercise-induced muscle injury. Here, we developed a carefully monitored and individualized neuromuscular electrical stimulation (NMES) training protocol of the mouse plantar flexor muscles. Each NMES training session consisted of 80 isometric contractions at a submaximal mechanical intensity corresponding to ~ 15% of maximal tetanic force to avoid muscle damage. NMES trained mice were stimulated for 2 x 3 consecutive days separated by one day of rest, for a total of 6 sessions. Experiments were conducted on C57BL/6J and BALB/c males at 10-12 weeks of age. NMES led to a robust myonuclear accretion and higher MuSC content in gastrocnemius muscle of both mouse lines, without overt signs of muscle damage/regeneration or muscle hypertrophy or force improvement. This new mouse model of myonuclear accretion relying on the main function of skeletal muscles, i.e., force production in response to electrical stimuli, will be of utmost interest to further understand the role of MuSCs in skeletal muscle adaptations.

BackgroundImmobilization, bed rest, or illness rapidly lead to weakness out of proportion to muscle atrophy. Although the contribution of muscle wasting to weakness is well described, the role of neuromuscular junction (NMJ) dysfunction in early disuse-related weakness is not well understood. ObjectiveWe investigated whether short-term unilateral hindlimb immobilization (HLI) in rats impairs NMJ transmission and contributes to muscle weakness out of proportion to atrophy. MethodsFour-month-old male Fischer-344/Brown Norway rats underwent 10 days of unilateral HLI (n=6) or remained mobile (n=6). Neuromuscular excitability and transmission were assessed using compound muscle action potentials (CMAP), repetitive nerve stimulation (RNS), and single-fiber electromyography (SFEMG). Muscle contractility testing quantified tetanic torque, and post-mortem analysis measured muscle mass. ResultsTen days of HLI reduced gastrocnemius, soleus, and plantaris muscle mass by [~]20-35%. Plantarflexion peak tetanic torque normalized to body weight declined by 23%, and torque-time integral was reduced by 36%, indicating disproportionate functional loss (muscle size versus contractile output) and supporting underlying neural impairment. CMAP amplitude decreased from 69 mV to 53.23 mV (a 22.9% reduction; p = 0.0109), indicating a loss of summated neuromuscular excitability. Furthermore, both RNS and SFEMG indicated features consistent with NMJ transmission defects. RNS revealed CMAP decrement from [~]0% pre-HLI to -9.15% at 40 Hz stimulation and -8.63% at 50 Hz post-HLI. Similarly, SFEMG confirmed marked NMJ transmission defects, with jitter increasing 103% and blocking increased from <1% to >11% of fibers. ConclusionsOur findings suggest that short-term immobilization produces rapid and pronounced impairments in NMJ transmission that contribute to weakness beyond the degree of muscle atrophy. These findings identify the NMJ as an early and vulnerable site of disuse-induced dysfunction and highlight the potential for synaptic-targeted therapies to preserve muscle performance during immobilization and recovery.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely recognized to potentially interfere with skeletal muscle regeneration. However, current knowledge is based almost exclusively on non-aspirin NSAIDs. Aspirin (ASA) differs from other NSAIDs in its ability to irreversibly acetylate cyclooxygenase-2 (COX-2), thereby redirecting its activity toward a lipoxygenase (LOX)-like function that enables the production of unique ASA-triggered specialized pro-resolving lipid mediators (AT-SPMs). Despite this, the potential impact of ASA on musculoskeletal tissue repair remains poorly understood. This study directly compared the effect of ASA against non-ASA NSAIDs on in vitro myogenesis and in vivo skeletal muscle injury and regeneration. Unlike non-ASA NSAIDs, including indomethacin (INDO), celecoxib, and SC-236, which markedly impaired C2C12 myotube formation at concentrations near their pharmacological ranges, ASA only interfered with myogenesis at overtly supraphysiological concentrations. In mice, an oral dose of 3 mg/kg/day INDO following barium chloride-induced muscle injury reduced regenerating myofiber cross-sectional area and impaired the recovery of muscle force-generating capacity. In contrast, a potency-matched oral treatment with 30 mg/kg/day ASA hastened the resolution of cellular inflammation, promoted myonuclear accretion, and improved recovery of absolute muscle strength. The beneficial effects of ASA on inflammatory resolution and muscle strength--but notably not myonuclear accretion--were reversed in mice co-treated with ASA + INDO. These findings demonstrate that, unlike non-ASA NSAIDs, ASA does not impair skeletal muscle regeneration and may promote a favorable early inflammatory environment for repair via unique COX-dependent pro-resolving and COX-independent anabolic mechanisms.

Residual force enhancement (rFE), defined as increased isometric force following active lengthening compared to a fixed-end isometric contraction at the same muscle length and level of activation, is present across all scales of muscle. While rFE is always present at the cellular level, often rFE non-responders are observed during joint-level voluntary contractions. We compared rFE between the joint level and single fibre level (vastus lateralis biopsies) in 16 young males. In-vivo voluntary knee-extensor rFE was measured by comparing steady-state isometric torque between a stretch-hold (maximal activation at 150{degrees}, stretch to 70{degrees}, hold) and a fixed-end isometric contraction, with ultrasonographic recording of vastus lateralis fascicle length (FL). Fixed-end contractions were performed at 67.5{degrees}, 70{degrees}, 72.5{degrees}, and 75{degrees}; the joint angle that most closely matched FL of the stretch-hold contractions isometric steady-state was used to calculate rFE. The starting and ending FLs of the stretch-hold contraction were expressed as % optimal FL, determined via torque-angle relationship. In single fibre experiments, the starting and ending fibre lengths were matched relative to optimal length determined from in-vivo testing, yielding an average sarcomere excursion of [~]2.2-3.4{micro}m. There was a greater magnitude of rFE at the single fibre ([~]20%) than joint level ([~]5%) (P=0.004), with non-responders only observed at the joint level. By comparing rFE across scales within the same participants, we show the development of the rFE non-responder phenomenon is upstream of rFEs cellular mechanisms, with rFE only lost rather than gained when scaling from single fibres to the joint level.

Mass is a fundamental aspect of muscle contractile function, yet the inertial effects of inactive muscle mass is generally neglected in modeling and not quantified in studies on small muscles or isolated fibers. However, during submaximal contractions, inactive muscle tissue may take longer to be accelerated by active fibers, and may be subject to prolonged deceleration, both of which may potentially reduce force development and work output. We sought to test if inactive tissue mass imposes an inertial penalty on muscle performance, using in situ sinusoidal work-loop experiments on rat plantaris muscles. Regional fascicle dynamics, measured across supramaximal and submaximal levels of activation, showed that decreasing activation significantly reduced fascicle strain and increased both shortening and lengthening latency. Contrary to our predictions, however, reductions in work, beyond those explained by decreased fascicle strain, were negligible. Normalized work did not decline disproportionately relative to force, suggesting no clear inertial penalty on work at this muscle size. Our findings suggest that while inactive muscle mass influences the dynamics of submaximal contractions, its impact on work during submaximal contractions at small muscle sizes is limited.

Increased serial sarcomere number (SSN) has been observed in rats via downhill running training due to the emphasis on active lengthening contractions; however, little is known about the influence on dynamic contractile function. Therefore, we employed 4 weeks of weighted downhill running training in rats, then assessed soleus SSN and work loop performance. We hypothesized trained rats would produce greater net work output during faster, higher-strain work loops due to a greater SSN. Thirty-one Sprague-Dawley rats were assigned to a control or training group. Weight was added during downhill running via a custom-made vest, progressing from 5-15% body mass. Following sacrifice, the soleus was dissected, and a force-length relationship was constructed. Work loops (active shortening followed by passive lengthening) were then performed about optimal muscle length (LO) at 1.5-3-Hz cycle frequencies and 1-7-mm strains to assess net work output. Muscles were then fixed in formalin at LO. Fascicle lengths and sarcomere lengths were measured and used to calculate SSN. Intramuscular collagen content and crosslinking were quantified via a hydroxyproline content and pepsin-solubility assay. Trained rats had longer fascicle lengths (+13%), greater SSN (+8%), greater specific active forces (+50%), and lower passive forces (-45-62%) than controls (P<0.05). There were no differences in collagen parameters (P>0.05). Net work output was greater (+101-424%) in trained than control rats for the 1.5-Hz loops at 1, 3, and 5-mm strains (P<0.05) and showed relationships with fascicle length (R2=0.14-0.24, P<0.05). These results suggest training-induced longitudinal muscle growth may improve dynamic performance.

Whether and how ovarian hormone fluctuations mediate the skeletal muscle response to ageing in females remains to be elucidated. We examined a tightly controlled, cross-sectional cohort of 96 females between 18-80 years of age to map the functional and molecular trajectory of muscle ageing and determine its relationship with female sex hormones. Across every decade, we quantified body composition (using dual-energy x-ray absorptiometry), muscle morphology (using peripheral quantitative computed tomography), and voluntary and evoked muscle function. Circulating sex hormone concentrations were measured with gas chromatography mass spectrometry and immunoassays. Morphology and gene expression of vastus lateralis muscle samples were assessed with immunohistochemical staining and RNA sequencing, respectively. Age was negatively associated with muscle mass, strength, and muscle fibre size, and positively associated with hybrid type I/II fibre prevalence and fibrosis. We found 37 unique patterns of gene expression across individual decades of age. Immune signalling, cellular adhesion, and extracellular matrix organisation pathways were the most upregulated with age, while mitochondrial function pathways were the most downregulated. Independently of age, circulating oestradiol and progesterone, but not testosterone, concentrations were positively associated with lean mass and negatively associated with hybrid muscle fibres across the lifespan. Oestrogen receptor binding sites were significantly enriched in upregulated genes in pre- versus post-menopausal muscle, suggesting a reduction in the translation of oestrogen target genes after menopause. Altogether, sex hormone fluctuations across the female lifespan may contribute to age-related muscle wasting, although longitudinal and interventional studies are needed to determine the causal nature of the relationship. Abstract figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/25331955v2_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@15610c9org.highwire.dtl.DTLVardef@168104corg.highwire.dtl.DTLVardef@105ef53org.highwire.dtl.DTLVardef@a34522_HPS_FORMAT_FIGEXP M_FIG C_FIG This study mapped the trajectory of muscle ageing at the whole-body, whole-muscle, and cellular level in 96 healthy females aged between 18 and 80 years old, while controlling for confounding lifestyle factors. Muscle mass and function declined with age, concomitant to a reduction in type I fibre size and increase in hybrid type I/IIa fibres. Patterns of muscle gene expression were mapped across ageing, showing an increase in immune cell signalling and a decline in mitochondrial respiration pathways. Circulating sex hormones were significantly associated with muscle mass, morphology, and gene expression across the lifespan. Key points summary O_LIFemales live longer than males but experience worse disability in the later decades of life, highlighting the need to study female-specific patterns of ageing. C_LIO_LIThis study mapped female body composition, muscle morphology, function, and gene expression across every decade from 18 to 80 years of age in tightly controlled conditions and examined the relationships with circulating sex hormones. C_LIO_LIUnique patterns of muscle gene expression across ageing showed an overall increase in immune signalling and a decrease in mitochondrial respiration pathways, but limited associations with circulating sex hormones. C_LIO_LIIndependently of age, circulating oestradiol and progesterone, but not testosterone, were associated with muscle mass and morphology across the lifespan, after adjusting for influential lifestyle factors (protein intake and physical activity). C_LIO_LIFluctuations in female sex hormones across the lifespan should be considered when developing therapies to mitigate age-related muscle wasting and improve the female health span. C_LI

Blood flow restriction (BFR) with low-load exercise (BFR-exercise) is an increasingly popular tool used to increase muscle strength and attenuate muscle atrophy, especially after injury or surgery. However, the mechanisms underlying BFR-mediated muscle growth are not well understood. Likely contributing to the mechanistic knowledge gap, rodent models of BFR-exercise have not been well described. In this methods paper, we demonstrate a comprehensive, clinically relevant protocol to establish BFR-exercise in awake rats. This protocol includes generating a muscle loss state via bilateral ACL-R, determining targeted blood flow occlusion pressures, and performing weighted hind-limb knee extension exercises with BFR. These methods can be used for further application in mechanistic and physiologic studies of BFR-exercise.

The present study aimed to characterise the temporal recovery pattern of contralateral-homologous torque following a bout of unilateral resistance exercise (RE). Ten young, healthy, recreationally active, resistance-trained men performed 10 sets of 10 repetitions of knee extensor (KE) contractions at 50 % 1RM with 1 min rest between sets. Isometric maximal voluntary contraction (MVC) peak torque (PT), surface electromyography (sEMG), muscle soreness and serum creatine kinase (CK) levels were assessed immediately before and 5 min after RE cessation, and then +4 h, +24 h, +48 h and +72 h later. Data are presented as mean [95 % CI] % change from pre-exercise values. RE evoked a minor increase in CK and pain in the late recovery period (+24 h to +72 h) (P < 0.034) and decreases in ipsilateral KE PT were observed immediately post-exercise (-26 [-33, -18] %, P < 0.001) and up to +48 h (-12 [-19, -4] %, P = 0.006). Measurable decreases in PT were also observed in the non-exercised contralateral KE immediately post-exercise (-8 [-13, -3] %, P = 0.006) up to +24 h (-8 [-15, 0] %, P = 0.020), but were significantly lower than the ipsilateral KE PT (P < 0.05). These findings suggest the presence of crossover fatigue following RE in young, healthy, active, resistance-trained men, however, the magnitude and temporal recovery are substantially less severe and protracted in the contralateral homologous KE.

The contributions of intrinsic muscle fiber resistance during mechanical perturbations to standing and other postural behaviors are unclear. Muscle stiffness, a traditional metric for estimating muscles intrinsic resistance to stretch, is known to vary depending on the current level and history of the muscles activation, as well as the muscles recent movement history; this property has been referred to as history dependence or muscle thixotropy. However, we currently lack sufficient data about the degree to which muscle stiffness is modulated across posturally-relevant characteristics of muscle stretch and activation. Here, we characterized the history dependence of muscles resistance to stretch in single, permeabilized, activated, muscle fibers in posturally-relevant stretch conditions and activation levels. We used a classic paired muscle stretch paradigm, varying the amplitude of a "conditioning" triangular stretch-shorten cycle followed by a "test" ramp-and-hold imposed after a variable inter-stretch interval. We tested low (<15%), intermediate (15-50%) and high (>50%) muscle fiber activation levels, evaluating short-range stiffness and total impulse in the test stretch. Muscle fiber resistance to stretch remained high at conditioning amplitudes of <1% L0 and inter-stretch intervals of >1 s, characteristic of healthy standing postural sway. A ~70% attenuation of muscle resistance to stretch was reached at conditioning amplitudes of >3% L0 and inter-stretch intervals of <0.1s, characteristic of larger, faster postural sway in balance-impaired individuals. Overall, amplitude and inter-stretch interval interact to disrupt myofilaments such that intrinsic resistance to stretch is attenuated if the stretch is large enough and/or frequent enough. Summary StatementIntrinsic muscle fiber resistance to stretch is preserved after small, slow pre-movements based on healthy postural sway, but markedly reduced as pre-movements increase to emulate abnormal postural sway.

Exercise-induced hypoalgesia (EIH) is a transient decrease in pain perception that can be observed following various tasks, including non-painful low-intensity and painful high-intensity exercise. The application of blood flow occlusion (BFO) can help enhance exercise adaptations while being able to exercise at a low intensity, which has important implications for clinical and rehabilitative settings. Through descending inhibitory pathways, BFO-induced pain can potentially alleviate exercise-induced pain. This study aimed to assess whether the superimposition of BFO - and its associated augmented perceived responses - during low-intensity, low-volume resistance exercise could induce hypoalgesia. Nineteen healthy adults (10 females) attended three sessions: i) no exercise (CTRL), ii) two minutes of dynamic single-leg knee extension at 10% body weight (EXER), and iii) EXER with complete occlusion applied to the upper exercising leg (OCCL). Handheld algometry-derived pain pressure threshold (PPT) of the trapezius and contralateral and ipsilateral rectus femoris muscles were measured pre- and post-exercise, and after 5 and 10 min of recovery. Perceived pain (0-10) and effort (6-20) were also rated after exercise. Although pain and effort were augmented in the OCCL condition (Pain: 6{+/-}2; Effort: 14{+/-}3) compared to CTRL (Pain: 2{+/-}2, p<0.001; Effort 9{+/-}2, p=0.017), PPT of all muscles did not change across time nor between any conditions. Therefore, the low-intensity, low-volume resistance exercise prescribed in the present study was insufficient to evoke EIH even with the application of BFO-induced pain.

The myosin-containing thick filament has recently been shown to alter its resting activation level in response to multiple diseases and therapeutics. Changes in thick filament resting activation level are caused by myosin heads transitioning between OFF and ON conformational states. Functionally, this modulation of thick filament activation level is a key regulatory step in muscle contraction and a promising therapeutic target. The availability of resting ON-state myosin heads governs dynamic contractility, which is critical to physical function and well-being. At present, there is a lack of compounds favouring this ON-state in resting skeletal muscle. Piperine is a molecule known to bind to myosin and increase submaximal isometric contractility in fast and slow skeletal muscle. Yet, effects on dynamic contractility and the underlying mechanism responsible for the observed effects in skeletal muscles remain unclear. Here, we used fibre small-angle X-ray diffraction and intact-muscle ex vivo contractility experiments to determine the effects of piperine on resting myosin structure and dynamic contractility in fast and slow rat muscles. X-ray diffraction data suggest that piperine promotes an OFF-to-ON transition of myosin in resting skeletal muscle, increasing the availability of myosin heads for force generation. Functionally, piperine substantially enhanced dynamic contractility in both muscle types, with greater improvements in slow muscle during maximal activation. These findings establish piperine as a tool to probe thick-filament activation in skeletal muscle, highlighting fibre-type-specific effects of thick-filament activation on the recruitment of the contractile reserve capacity. Key Points- Piperine is a compound known to bind to skeletal muscle myosin and enhance isometric contractility in fast and slow muscles, but the underlying molecular mechanisms and effects on dynamic contractile function remain unknown. - We show that piperine increases the activation level of the myosin-containing thick filament in resting fast and slow skeletal muscle, which may explain the effect of piperine on contractile function. - Piperine substantially increases the maximal contractile power of both fast and slow skeletal muscles at low-frequency activation; however, it only enhances the maximal power in slow skeletal muscle at high-frequency activation. - Our data reveal potentiation of dynamic contractility with fibre-type-dependent magnitudes in response to piperine-induced activation of the resting thick filament, a phenomenon that requires further investigation and may ultimately be exploited in the treatment of diseases characterised by muscle weakness. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/689918v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1ff3672org.highwire.dtl.DTLVardef@4f809forg.highwire.dtl.DTLVardef@1857404org.highwire.dtl.DTLVardef@83d958_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Abstract figure legend: We investigated the effects of piperine on 1) the activation level of the resting thick filament and 2) dynamic contractility in fibres and intact slow (soleus) and fast (extensor digitorum longus, EDL) rat muscles, respectively. The activation level of the resting thick filament was assessed pre- and post-piperine incubation using small-angle X-ray diffraction. Dynamic contractility was assessed at submaximal and maximal activation levels by constructing low- and high-frequency force-velocity curves and corresponding power curves using an ex vivo contraction setup. The setup allows for simultaneous experiments on the effects of piperine and vehicle treatment in contralateral muscles. We found that piperine increased the activation level of the resting thick filament by favouring the ON-myosin state in fibres from both muscle types. In whole muscle preparations, piperine also induced substantial increases in dynamic contractility, especially in slow soleus muscle. C_FIG

AimDespite men exhibiting greater muscle strength and fatigibility than women, it remains unclear if there are sex-based differences in muscle recruitment strategies e.g. motor unit (MU) recruitment and modulation of firing rate (FR) at normalised forces and during progressive increases in force. MethodsTwenty-nine healthy male and thirty-one healthy female participants (18-35 years) were studied. Intramuscular electromyography was used to record individual motor unit potentials (MUPs) and near fibre MUPs from the vastus lateralis (VL) during 10% and 25% maximum isometric voluntary contractions (MVC), and spike-triggered averaging was used to obtain motor unit number estimates (MUNE) of the VL. Multilevel mixed-effects linear regression models were used to investigate the effects of sex at each contraction level. ResultsMen exhibited greater muscle strength (p<0.001) and size (p<0.001) than women, with no difference in force steadiness at 10% or 25% MVC. Women had smaller MUs and higher FR at 10% MVC (both p<0.02), similar to that at 25% MVC in MU size (p=0.062) and FR (p=0.031). However, both sexes showed similar increases in MU size and FR when moving from low-to mid-level contractions. There were no sex differences in any near fibre MUP parameters or in MUNE. ConclusionIn the vastus lateralis, women produce muscle force via different neuromuscular recruitment strategies to men which is characterised by smaller MUs discharging at higher rates. However, similar strategies are employed to increase force production from low to moderate contractions. These findings of similar proportional increases between sexes support the use of mixed sex cohorts in studies of this nature. Key pointsO_LIIncreases in muscle force production are mediated by motor unit (MU) recruitment, and MU firing rate (FR). C_LIO_LIWomen are underrepresented in studies of human neuromuscular research and markedly differ to men in a number of aspects of neuromuscular function, yet little is known of the recruitment strategies of each. C_LIO_LIHere we demonstrate men and women have similar vastus lateralis MU number estimates, yet women recruit smaller MUs with higher FR than men at normalised contraction levels. However, increases in force are achieved via similar trajectories of MU recruitment and MU FR in men and women. C_LIO_LIAlthough men and women exhibit divergent neuromuscular recruitment strategies to achieve normalised forces, increases in force are achived similarly and support the inclusion of mixed sex cohorts in studies of this nature. C_LI

Intracellular accumulation of hydrogen ions (H+) and inorganic phosphate (Pi) have temperature-dependent effects on single fiber contractile function between 10-30{degrees}C. In vivo, human skeletal muscle temperatures range between 35-38{degrees}C, and although contractile function is highly dependent on temperature, the effects of fatigue-inducing [H+] and [Pi] on contractile mechanics at 37{degrees}C is unknown. Using sinusoidal analysis, the independent and combined effects of these metabolites on cellular and molecular contractile function were determined at 37{degrees}C in slow-contracting myosin heavy chain (MHC) I and fast-contracting MHC IIA fibers from vastus lateralis muscle of 13 older adults (8 females), under four conditions: maximal calcium activation ("control"; 5 mM Pi, pH 7.0), high Pi (30 mM), low pH (6.2), and fatigue (30 mM Pi and pH 6.2). Specific tension (force/cross-sectional area, mN/mm2) in both fiber types was reduced only under fatigue conditions (20-26%). MHC I fibers had slower cross-bridge kinetics with fewer or less stiff strongly-bound myosin-actin cross-bridges in high Pi, low pH, and fatigue. In contrast, fatigued MHC IIA fibers had faster cross-bridge kinetics with increased myofilament and/or cross-bridge viscosity. Single fiber oscillatory work was reduced in both fiber types when Pi or pH alone was altered. However, fatigue conditions returned oscillatory work values toward control through alterations to cross-bridge kinetics in MHC I fibers and changes to work absorption and production processes in MHC IIA fibers. These findings quantify fiber-type specific mechanical and kinetic mechanisms of fatigue in human skeletal muscle at 37{degrees}C, thus advancing our understanding of metabolite-based muscle fatigue in vivo. KEY POINTS SUMMARYO_LIWorking skeletal muscle increases intracellular concentrations of hydrogen ion and inorganic phosphate, leading to fatigue, or loss of force-generating capacity C_LIO_LITemperature plays a well-established role in the muscle response to hydrogen ion and/or inorganic phosphate accumulation, but has not previously been studied at human body temperature (37{degrees}C) C_LIO_LIAt 37{degrees}C, reduced force generation only occurs when high phosphate and hydrogen ions are combined, not when changed individually C_LIO_LIIn slow-contracting fibers, fatigue slowed myosin-actin cross-bridge kinetics and reduced the number or stiffness of strongly-bound cross-bridges. In fast-contracting fibers, fatigue increased myosin-actin cross-bridge kinetics and increased myofilament viscosity. C_LIO_LIThe distinct responses by fiber type to fatigue provides new insight into its mechanisms and advances our understanding of the whole muscle and body responses to fatigue C_LI Abstract Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/672942v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@e4ece3org.highwire.dtl.DTLVardef@17c62eforg.highwire.dtl.DTLVardef@1434e30org.highwire.dtl.DTLVardef@1c2446c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Age-related reductions in muscle fiber size and contractile function, particularly in fibers expressing fast myosin heavy chains, contribute to declines in whole-muscle power. However, methodological limitations in estimating fiber size during contractile experiments have likely contributed to conflicting findings regarding whether reduced single-fiber force and power in older adults reflects their smaller size and/or impaired intrinsic contractile function. To address this, we coupled single-fiber contractile experiments with 3D-imaging in 7 young (19-40yrs) and 6 older (69-84yrs) males to assess intrinsic contractile function and compare agreement between 3D-derived cross-sectional area (CSA) and CSA estimates obtained either in air or solution. Fast fiber CSA from older males were [~]28-45% smaller across measurement conditions compared with young, whereas slow fiber CSA did not differ. Accordingly, absolute force and power of fast fibers were 41% and 37% lower. When normalized to CSA from measurements in air or 3D-imaging, size-specific force and power either did not differ or were greater in older adults, indicating preserved intrinsic contractile function in both fiber types. This was supported by no age-related differences in the rate of tension redevelopment (ktr), a size-independent measure of intrinsic contractile function. In contrast, size-specific force and power calculated using solution-based CSA estimates were lower in older compared with young adults, and Bland-Altman analyses demonstrated the poorest agreement between solution-based and 3D CSA measurements. These findings indicate that intrinsic contractile function is preserved with aging and suggest that methodological differences in CSA measurement contributes to the disparate findings in the literature.

Skeletal muscle hypertrophy results from the integrated regulation of anabolic and proteolytic processes in response to mechanical loading. Although increases in resistance training (RT) volume are used to increase mechanical stress, it remains uncertain whether large and abrupt volume progressions could exceed muscle adaptive capacity by disrupting the balance between anabolic and catabolic signaling. The present study investigated whether a large increase in weekly RT volume (+120%) leads to impaired hypertrophic outcomes and intracellular regulatory responses compared with a modest increase (+20%). Twenty-five resistance-trained men and women (18-35 years old) completed an 8-week randomized, single-blind, within-subject unilateral intervention. Each participant trained both legs twice weekly, with one leg assigned to the large (VOL120) and the contralateral leg to the modest (VOL20) weekly volume progressions relative to habitual training volume. Vastus lateralis muscle cross-sectional area (mCSA) was assessed by ultrasonography before and after training. Muscle biopsies were obtained at baseline, post-intervention, and 24 h after the last session to quantify muscle fiber cross-sectional area (fCSA), satellite cell myonuclear content, and anabolic/catabolic signaling markers. Both protocols induced increases in mCSA over time (p<0.001), with no protocol vs. time interaction. No significant effects were observed for fCSA nor satellite cell number or myonuclear content. Additionally, molecular responses related to translational regulation and protein degradation were largely similar between protocols. Collectively, these data indicate that a large, abrupt increase in weekly set volume does not impair hypertrophic adaptations or meaningfully alter the anabolic-catabolic signaling profile in resistance-trained individuals.