Skeletal Muscle Force Production Research Articles

Decoding the forces that shape muscle stem cell function.

Skeletal muscle is a force-producing organ composed of muscle tissues, connective tissues, blood vessels, and nerves, all working in synergy to enable movement and provide support to the body. While robust biomechanical descriptions of skeletal muscle force production at the body or tissue level exist, little is known about force application on microstructures within the muscles, such as cells. Among various cell types, skeletal muscle stem cells reside in the muscle tissue environment and play a crucial role in driving the self-repair process when muscle damage occurs. Early evidence indicates that the fate and function of skeletal muscle stem cells are controlled by both biophysical and biochemical factors in their microenvironments, but much remains to accomplish in quantitatively describing the biophysical muscle stem cell microenvironment. This book chapter aims to review current knowledge on the influence of biophysical stresses and landscape properties on muscle stem cells in heath, aging, and diseases.

Skeletal muscle-specific inducible AMPKα1/α2 knockout mice develop muscle weakness, glycogen depletion, and fibrosis that persists during disuse atrophy.

The 5' adenosine monophosphate-activated protein kinase (AMPK) is an important skeletal muscle regulator implicated as a possible therapeutic target to ameliorate the local undesired deconditioning of disuse atrophy. However, the muscle-specific role of AMPK in regulating muscle function, fibrosis, and transcriptional reprogramming during physical disuse is unknown. The purpose of this study was to determine how the absence of both catalytic subunits of AMPK in skeletal muscle influences muscle force production, collagen deposition, and the transcriptional landscape. We generated skeletal muscle-specific tamoxifen-inducible AMPKα1/α2 knockout (AMPKα-/-) mice that underwent 14 days of hindlimb unloading (HU) or remained ambulatory for 14 days (AMB). We found that AMPKα-/- during ambulatory conditions altered body weight and myofiber size, decreased muscle function, depleted glycogen stores and TBC1 domain family member 1 (TBC1D1) phosphorylation, increased collagen deposition, and altered transcriptional pathways. Primarily, pathways related to cellular senescence and mitochondrial biogenesis and function were influenced by the absence of AMPKα. The effects of AMPKα-/- persisted, but were not worsened, following hindlimb unloading. Together, we report that AMPKα is necessary to maintain skeletal muscle quality.NEW & NOTEWORTHY We determined that skeletal muscle-specific AMPKα knockout (KO) mice display functional, fibrotic, and transcriptional alterations before and during muscle disuse atrophy. We also observed that AMPKα KO drives muscle fibrosis and pathways related to cellular senescence that continues during the hindlimb unloading period.

Impact of Human Recombinant Irisin on Tissue-Engineered Skeletal Muscle Structure and Function.

Tissue engineering of exogenous skeletal muscle units (SMUs) through isolation of muscle satellite cells from muscle biopsies is a potential treatment method for acute volumetric muscle loss (VML). A current issue with this treatment process is the limited capacity for muscle stem cell (satellite cell) expansion in cell culture, resulting in a decreased ability to obtain enough cells to fabricate SMUs of appropriate size and structural quality and that produce native levels of contractile force. This study determined the impact of human recombinant irisin on the growth and development of three-dimensional (3D) engineered skeletal muscle. Muscle satellite cells were cultured without irisin (control) or with 50, 100, or 250 ng/mL of irisin supplementation. Light microscopy was used to analyze myotube formation with particular focus placed on the diameter and density of the monotubes during growth of the 3D SMU. Following the formation of 3D constructs, SMUs underwent measurement of maximum tetanic force to analyze contractile function, as well as immunohistochemical staining, to characterize muscle structure. The results indicate that irisin supplementation with 250 ng/mL significantly increased the average diameter of myotubes and increased the proliferation and differentiation of myoblasts in culture but did not have a consistent significant impact on force production. In conclusion, supplementation with 250 ng/mL of human recombinant irisin promotes the proliferation and differentiation of myotubes and has the potential for impacting contractile force production in scaffold-free tissue-engineered skeletal muscle.

Sex differences in isolated skeletal muscle force production: a spectrum of responses

Whether sex differences in skeletal muscle microvascular responses differ in response to muscle contraction is currently unknown. To determine vascular responses to muscle contraction in vivo we use intravital microscopy using thin skeletal muscle models of mixed fibre types (i.e. hamster retractor muscle). However, to effectively quantify this relationship in vivo, the initial muscle contraction stimulus must be constant across the sexes and thus, we must first determine whether there are sex differences in skeletal muscle force generation. We hypothesized that sexual dimorphisms in force production would not be apparent in muscles of any fibre type composition. To test this hypothesis, we used skeletal muscles with unique fibre type characteristics and oxidative capacities. Mouse soleus (SOL: primarily type 1 >75%), extensor digitorum longus (EDL: primarily type IIB ~60%), and costal diaphragm (DIA: primarily type IIA ~55%) muscles were isolated from adult male and female CD-1 mice. The retractor muscle (RET: primarily type IIX ~58%) was also isolated from adult male and female Syrian hamsters. Muscles were electrically stimulated in vitro and force production was quantified at optimal length during a force-frequency test (1-120Hz) and twitch (1Hz) contractions. In mice, females generated greater force during the force-frequency test in SOL (10-120Hz) and EDL (30-120Hz) and generated greater twitch forces compared to males (SOL: female 34.7 ± 2.4 mN/mm 2 , n=21 vs male 27.8 ± 2.0 mN/mm 2 , n=17; EDL: female 41.2 ± 2.2 mN/mm 2 , n=35 vs male 32.5 ± 2.1 mN/mm 2 , n=29). Females generated greater force during the force frequency test in DIA at 30Hz only and no significant sex differences were observed in DIA twitch force (female 0.24 ± 0.02 g/mg, n=19 vs male 0.20 ± 0.02 g/mg, n=21). In hamster RET no significant sex differences in force were observed during the force frequency test or during the twitch contraction (female 0.13 ± 0.01 g/mg, n=19 vs male 0.14 ± 0.02 g/mg, n=20). Collectively, these findings demonstrate that sex differences in force production were observed in muscles primarily comprised of slow oxidative (type 1) and fast glycolytic (type IIB) fibres but not in muscles primarily comprised of intermediate fibres (type IIA and type IIX). These data show that on a spectrum of muscle fibres from slow oxidative to fast glycolytic, sex differences are most apparent on the extremes of the spectrum, whereas sexual dimorphisms in the middle of the spectrum are not apparent. This work is supported by NSERC. NSERC This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Intermittent treatment with elamipretide preserves exercise tolerance in aged female mice.

The pathology of aging impacts multiple organ systems, including the kidneyand skeletal and cardiac muscles. Long-term treatment with the mitochondrial-targeted peptide elamipretide has previously been shown to improve in vivo mitochondrial function in aged mice, which is associated with increased fatigue resistance and treadmill performance, improved cardiovascular diastolic function, and glomerular architecture of the kidney. However, elamipretide is a short tetrameric peptide that is not orally bioavailable, limiting its routes of administration. This study tested whether twice weekly intermittent injections of elamipretide could recapitulate the same functional improvements as continuous long-term infusion. We found that intermittent treatment with elamipretide for 8 months preserved exercise tolerance and left ventricular mass in mice with modest protection of diastolic function and skeletal muscle force production but did not affect kidney function as previously reported using continuous treatment.

Effects of hydroxyurea on skeletal muscle energetics and force production in a sickle cell disease murine model.

Hydroxyurea (HU) is commonly used as a treatment for patients with sickle cell disease (SCD) to enhance fetal hemoglobin production. This increased production is expected to reduce anemia (which depresses oxygen transport) and abnormal Hb content alleviating clinical symptoms such as vaso-occlusive crisis and acute chest syndrome. The effects of HU on skeletal muscle bioenergetics in vivo are still unknown. Due to the beneficial effects of HU upon oxygen delivery, improved skeletal muscle energetics and function in response to a HU treatment have been hypothesized. Muscle energetics and function were analyzed during a standardized rest-exercise-recovery protocol, using 31P-magnetic resonance spectroscopy in Townes SCD mice. Measurements were performed in three groups of mice: one group of 2-mo-old mice (SCD2m, n = 8), another one of 4-mo-old mice (SCD4m, n = 8), and a last group of 4-mo-old mice that have been treated from 2 mo of age with HU at 50 mg/kg/day (SCD4m-HU, n = 8). As compared with SCD2m mice, SCD4m mice were heavier and displayed a lower acidosis. As lower specific forces were developed by SCD4m compared with SCD2m, greater force-normalized phosphocreatine consumption and oxidative and nonoxidative costs of contraction were also reported. HU-treated mice (SCD4m-HU) displayed a significantly higher specific force production as compared with untreated mice (SCD4m), whereas muscle energetics was unchanged. Overall, our results support a beneficial effect of HU on muscle function.NEW & NOTEWORTHY Our results highlighted that force production decreases between 2 and 4 mo of age in SCD mice thereby indicating a decrease of muscle function during this period. Of interest, HU treatment seemed to blunt the observed age effect given that SCD4m-HU mice displayed a higher specific force production as compared with SCD4m mice. In that respect, HU treatment would help to maintain a higher capacity of force production during aging in SCD.

Titin force in muscle cells alters lattice order, thick and thin filament protein formation

Skeletal muscle force production is increased at longer compared to shorter muscle lengths because of length-dependent priming of thick filament proteins in the contractile unit before contraction. Using small-angle X-ray diffraction in combination with a mouse model that specifically cleaves the stretch-sensitive titin protein, we found that titin cleavage diminished the length-dependent priming of the thick filament. Strikingly, a titin-sensitive, length-dependent priming was also present in thin filaments, which seems only possible via bridge proteins between thick and thin filaments in resting muscle, potentially myosin-binding protein C. We further show that these bridges can be forcibly ruptured via high-speed stretches. Our results advance a paradigm shift to the fundamental regulation of length-dependent priming, with titin as the key driver.

High-intensity Interval Training On Muscle Strength And Rate Of Torque Development In Female Lacrosse Players.

Sports, which include lacrosse, require sudden directional changes and rapid alterations in pace both of which are highly dependent on explosive skeletal muscle force production. Previous research has shown that high-intensity interval training (HIIT) increases maximal sprint speed, muscle power, and overall performance in field sports. Limited research on the potential of HIIT to impact skeletal muscle strength and rate of torque development in lacrosse players exist. PURPOSE: To determine the effects of HIIT in combination with lacrosse specific training on lower-limb skeletal muscle strength and rate of torque development in female high school lacrosse players (15 ± 2 yrs.). METHODS: Fourteen healthy female lacrosse players from a local high school performed HIIT three times per week in addition to their specific lacrosse training program for 11 weeks (preseason plus competitive season). Right lower-limb skeletal muscle performance was assessed Pre, Mid, and Post-HIIT training with concentric knee extensor contractions at three angular velocities (0, 1.6, and 3.1 rad·s-1). Outcome measures included: peak torque (Nm), time-to-peak torque (s), and rate of torque development (Nm·s-1). RESULTS: Isometric and concentric (3.1 rad·s-1) strength increased across the HIIT and lacrosse specific training period by 20% and 10%, respectively (p < 0.05). Combined training had no influence on time-to-peak torque (Pre: 2.2 ± 0.9 s; Post: 2.6 ± 0.5 s; p > 0.05). Rate of torque development for the first 50 ms of contraction increased by 42% at the mid-point [Pre: 607.3 ± 451.1; Mid: 862.7 ± 292.7 Nm·s-1, p < 0.05) and remained elevated at the Post-HIIT assessment (Post: 864.7 ± 255.4 Nm·s-1). No change in rate of torque development was observed over the 100-200 ms period of contraction. CONCLUSIONS: Combined HIIT with lacrosse specific training improved both static and dynamic knee extensor strength and enhanced rate of torque development within the first 50 ms of contraction in female high school lacrosse players. The inclusion of HIIT to standard pre-season and competitive lacrosse season training demonstrated no negative effects on lower-limb skeletal muscle performance.

Iron Supplementation Improves Skeletal Muscle Contractile Properties in Mice with CKD.

Patients with chronic kidney disease (CKD) frequently have compromised physical performance, which increases their mortality; however, their skeletal muscle dysfunction has not been characterized at the single-fiber and molecular levels. Notably, interventions to mitigate CKD myopathy are scarce. The effect of CKD in the absence and presence of iron supplementation on the contractile function of individual skeletal muscle fibers from the soleus and extensor digitorum longus muscles was evaluated in 16-week-old mice. CKD was induced by the adenine diet, and iron supplementation was by weekly iron dextran injections. Maximally activated and fatigued fiber force production was decreased 24%-52% in untreated CKD, independent of size, by reducing strongly bound myosin/actin cross-bridges and/or decreasing myofilament stiffness in myosin heavy chain (MHC) I, IIA, and IIB fibers. Additionally, myosin/actin interactions in untreated CKD were slower for MHC I and IIA fibers and unchanged or faster in MHC IIB fibers. Iron supplementation improved anemia and did not change overall muscle mass in CKD mice. Iron supplementation ameliorated CKD-induced myopathy by increasing strongly bound cross-bridges, leading to improved specific tension, and/or returning the rate of myosin/actin interactions toward or equivalent to control values in MHC IIA and IIB fibers. Skeletal muscle force production was significantly reduced in untreated CKD, independent of fiber size, indicating that compromised physical function in patients is not solely due to muscle mass loss. Iron supplementation improved multiple aspects of CKD-induced myopathy, suggesting that timely correction of iron imbalance may aid in ameliorating contractile deficits in CKD patients.

Sex Differences in Skeletal Muscle Force Production, Fatiguability and Recovery in Slow and Fast Twitch Muscles

Sex differences in skeletal muscle fatiguability have been well documented during isometric contractions in humans but whether this sexual dimorphism occurs in skeletal muscle at the cellular level is not well understood. Therefore, we aimed to determine if sex differences persisted in skeletal muscle function in isolated tissue. We hypothesized that sexual dimorphisms would not be apparent in twitch and tetanic contraction characteristics or fatigue and recovery of slow and fast twitch muscles. Soleus (SOL) and extensor digitorum longus (EDL) muscles were isolated from male and female CD‐1 mice. Muscle force production at optimal length was measured during 5 protocols: 1) force‐frequency relationship (1‐120Hz), 2) twitch contraction (1Hz), 3) non‐fatiguing contraction protocol (1 contraction/90sec, for 22.5 mins at submaximal (SOL‐25Hz; EDL‐50Hz) and maximal (SOL‐80Hz; EDL‐120Hz) stimulus frequencies), 4) fatiguing contraction protocols at submaximal and maximal stimulus frequencies using contraction frequencies of 15, 30, 45 and 60 contractions per min (cpm) over 5 mins and 5) recovery protocols (1contraction/90sec for 30 mins). The force‐frequency relationship revealed that females generated significantly larger force compared to males at stimulation frequencies of 10‐120Hz (SOL) and 30‐120Hz (EDL). Female SOL and EDL produced significantly higher forces during twitch contractions compared to males (SOL: male 27.8+/‐2.0 mN/mm2 vs. female 34.7+/‐2.4 mN/mm2; EDL: male 32.5+/‐2.1 mN/mm2 vs. female 41.2+/‐2.2 mN/mm2). No significant differences in time to peak contraction or time to half relaxation during the twitch were found between males and females. There were no differences in fatigue rates between male and female SOL or EDL during the non‐fatiguing contraction protocols. Fatiguing contraction protocols revealed that females were less fatiguable than males at submaximal stimulus frequencies at 30 and 45cpm (SOL) and at maximal stimulus frequencies at 45cpm (EDL). No significant differences in fatigue between males and females were found in the remaining 13 fatiguing contraction protocols. Significant differences between males and females in recovery protocols were found in only 4 of the 16 recovery protocols. Female SOL had less recovery of force compared to males at 25Hz at 30 and 45cpm, and at 80Hz at 45 and 60cpm. Collectively, these data demonstrate that there are sexual dimorphisms in absolute force production during twitch and a range of tetanic contractions in slow and fast twitch muscles. Although there were specific contraction parameters that showed enhanced fatigue resistance of females and less recovery of female slow twitch muscles, these findings were not systematic and not present in the vast majority of the fatigue/recovery protocols. Our data show that force production and fatiguability at the in vitro, cellular level cannot explain the in vivo observations in the literature and the lack of sexual dimorphisms present during the majority of fatigue/recovery protocols in vitro indicate that systemic influences may be driving these documented differences at the whole‐body level.

Prior Alcohol Use does not Enhance Skeletal Muscle Atrophy and Force Decrements Induced by Cancer Cachexia

Introduction/objectiveAlcohol (EtOH) is a well‐known myotoxin with up to 60% of alcoholics suffering from alcoholic myopathy, characterized by progressive weakening and atrophy of proximal muscles. EtOH consumption is also associated with increased risk of cancer and continued EtOH intake enhances a primary comorbidity of cancer, cancer cachexia, or the loss of skeletal muscle strength and mass. However, the effects of prior EtOH use on cancer cachexia remains unknown. Therefore, the study objective was to determine if the cessation of EtOH prior to cancer development influences skeletal muscle force production, fatigability, and twitch characteristics.HypothesisWe hypothesize that discontinuing EtOH use prior to cancer progression will have no effect on skeletal muscle force production in tumor bearing mice.MethodsMale CD2F1 mice acclimated to a non‐EtOH containing Lieber DeCarli liquid diet for one week. Then, the concentration of EtOH was increased to 20% EtOH kcal/day for 6 weeks in EtOH mice. Mice then returned to non‐EtOH containing diet and C26 colon cancer cells were inoculated into the right flank of cancer groups. After 2‐3 weeks of cancer development muscle function was assessed. Mice were anesthetized, with the distal tendon of the lower leg attached to a servomotor, stimulating the posterior crural muscle complex by percutaneous electrodes. Peak isometric force was taken at 100 Hz, while fatigability was assessed using 60 isometric contractions produced over 4‐mins given at 100 Hz, and fatigue recovery measured using 100 Hz stimulations at 5‐ and 10‐minutes later. Triceps surae complex was excised and weighed following the force protocol.ResultsCancer reduced triceps surae muscle weight in both control and EtOH consuming mice (p &lt; 0.0001). Cancer reduced force production (p= 0.009) of 100 Hz tetanic stimulation while no further effect of EtOH was observed. Main effects of C26 (p&lt; 0.0001) were observed for fatigue within both diets, revealing reduced force production and increased fatigue. Force production was decreased across the fatigue protocol in all groups (p&lt; 0.0001), while main effects of C26 (p&lt;0.0001) were observed within the control and EtOH diets, revealing similarly reduced force production and increased fatigue regardless of EtOH status. Fatigue recovery was also similar across all groups. Twitch half relaxation was increased in C26 mice (p= 0.0283), showing a disruption in relaxation that is cancer dependent and not exacerbated by EtOH. No changes in twitch maximum force or rate of force development were noted between groups.ConclusionsTumor‐bearing mice produced less force during a tetanic stimulation and fatigue protocol, in accordance with significant muscle atrophy of the triceps surae. The prior consumption of EtOH did not worsen cancer‐induced deficits in skeletal muscle contraction. As it has been shown previously that the continuation of EtOH throughout the progression of cancer enhances cancer cachexia, these data exemplify the clinical benefits of stopping EtOH consumption as early as possible.

Maximal strength training-induced increase in efferent neural drive is not reflected in relative protein expression of SERCA

IntroductionMaximal strength training (MST), performed with heavy loads (~ 90% of one repetition maximum; 1RM) and few repetitions, yields large improvements in efferent neural drive, skeletal muscle force production, and skeletal muscle efficiency. However, it is elusive whether neural adaptations following such high intensity strength training may be accompanied by alterations in energy-demanding muscular factors.MethodsSixteen healthy young males (24 ± 4 years) were randomized to MST 3 times per week for 8 weeks (n = 8), or a control group (CG; n = 8). Measurements included 1RM and rate of force development (RFD), and evoked potentials recordings (V-wave and H-reflex normalized to M-wave (M) in the soleus muscle) applied to assess efferent neural drive to maximally contracting skeletal muscle. Biopsies were obtained from vastus lateralis and analyzed by western blots and real-time PCR to investigate the relative protein expression and mRNA expression of Sarcoplasmic Reticulum Ca2+ ATPase (SERCA) 1 and SERCA2.ResultsSignificant improvements in 1RM (17 ± 9%; p < 0.001) and early (0–100 ms), late (0–200 ms) and maximal RFD (31–53%; p < 0.01) were observed after MST, accompanied by increased maximal Vmax/Msup-ratio (9 ± 14%; p = 0.046), with no change in H-reflex to M-wave ratio. No changes were observed in the CG. No pre- to post-training differences were found in mRNA or protein expressions of SERCA1 and SERCA2 in either group.ConclusionMST increased efferent neural drive to maximally contracting skeletal muscle, causing improved force production. No change was observed in SERCA expression, indicating that responses to high intensity strength training may predominantly be governed by neural adaptations.

Assessing the Potential of Nutraceuticals as Geroprotectors on Muscle Performance and Cognition in Aging Mice.

Aging and frailty are associated with a decline in muscle force generation, which is a direct consequence of reduced muscle quantity and quality. Among the leading contributors to aging is the generation of reactive oxygen species, the byproducts of terminal oxidation. Their negative effects can be moderated via antioxidant supplementation. Krill oil and astaxanthin (AX) are nutraceuticals with a variety of health promoting, geroprotective, anti-inflammatory, anti-diabetic and anti-fatigue effects. In this work, we examined the functional effects of these two nutraceutical agents supplemented via pelleted chow in aging mice by examining in vivo and in vitro skeletal muscle function, along with aspects of intracellular and mitochondrial calcium homeostasis, as well as cognition and spatial memory. AX diet regimen limited weight gain compared to the control group; however, this phenomenon was not accompanied by muscle tissue mass decline. On the other hand, both AX and krill oil supplementation increased force production without altering calcium homeostasis during excitation-contraction coupling mechanism or mitochondrial calcium uptake processes. We also provide evidence of improved spatial memory and learning ability in aging mice because of krill oil supplementation. Taken together, our data favors the application of antioxidant nutraceuticals as geroprotectors to improve cognition and healthy aging by virtue of improved skeletal muscle force production.

Minocycline Treatment Reduces Mass and Force Output From Fast-Twitch Mouse Muscles and Inhibits Myosin Production in C2C12 Myotubes.

Minocycline, a tetracycline-class of antibiotic, has been tested with mixed effectiveness on neuromuscular disorders such as amyotrophic lateral sclerosis, autoimmune neuritis and muscular dystrophy. The independent effect of minocycline on skeletal muscle force production and signalling remain poorly understood. Our aim here is to investigate the effects of minocycline on muscle mass, force production, myosin heavy chain abundance and protein synthesis. Mice were injected with minocycline (40 mg/kg i.p.) daily for 5 days and sacrificed at day six. Fast-twitch EDL, TA muscles and slow-twitch soleus muscles were dissected out, the TA muscle was snap-frozen and the remaining muscles were attached to force transducer whilst maintained in an organ bath. In C2C12 myotubes, minocycline was applied to the media at a final concentration of 10 μg/mL for 48 h. In minocycline treated mice absolute maximal force was lower in fast-twitch EDL while in slow-twitch soleus there was an increase in the time to peak and relaxation of the twitch. There was no effect of minocycline treatment on the other contractile parameters measured in isolated fast- and slow-twitch muscles. In C2C12 cultured cells, minocycline treatment significantly reduced both myosin heavy chain content and protein synthesis without visible changes to myotube morphology. In the TA muscle there was no significant changes in myosin heavy chain content. These results indicate that high dose minocycline treatment can cause a reduction in maximal isometric force production and mass in fast-twitch EDL and impair protein synthesis during myogenesis in C2C12 cultured cells. These findings have important implications for future studies investigating the efficacy of minocycline treatment in neuromuscular or other muscle-atrophy inducing conditions.

Skeletal Muscle Function Is Dependent Upon BRCA1 to Maintain Genomic Stability.

Breast Cancer gene 1 (BRCA1) is a large, multifunctional protein that regulates a variety of mechanisms in multiple different tissues. Our work established that Brca1 is expressed in skeletal muscle and localizes to the mitochondria and nucleus. Here, we propose BRCA1 expression is critical for the maintenance of force production and mitochondrial respiration in skeletal muscle.

Histidine dipeptides are key regulators of excitation-contraction coupling in cardiac muscle: Evidence from a novel CARNS1 knockout rat model

Histidine-containing dipeptides (HCDs) are abundantly expressed in striated muscles. Although important properties have been ascribed to HCDs, including H+ buffering, regulation of Ca2+ transients and protection against oxidative stress, it remains unknown whether they play relevant functions in vivo. To investigate the in vivo roles of HCDs, we developed the first carnosine synthase knockout (CARNS1−/−) rat strain to investigate the impact of an absence of HCDs on skeletal and cardiac muscle function. Male wild-type (WT) and knockout rats (4 months-old) were used. Skeletal muscle function was assessed by an exercise tolerance test, contractile function in situ and muscle buffering capacity in vitro. Cardiac function was assessed in vivo by echocardiography and cardiac electrical activity by electrocardiography. Cardiomyocyte contractile function was assessed in isolated cardiomyocytes by measuring sarcomere contractility, along with the determination of Ca2+ transient. Markers of oxidative stress, mitochondrial function and expression of proteins were also evaluated in cardiac muscle. Animals were supplemented with carnosine (1.8% in drinking water for 12 weeks) in an attempt to rescue tissue HCDs levels and function. CARNS1−/− resulted in the complete absence of carnosine and anserine, but it did not affect exercise capacity, skeletal muscle force production, fatigability or buffering capacity in vitro, indicating that these are not essential for pH regulation and function in skeletal muscle. In cardiac muscle, however, CARNS1−/− resulted in a significant impairment of contractile function, which was confirmed both in vivo and ex vivo in isolated sarcomeres. Impaired systolic and diastolic dysfunction were accompanied by reduced intracellular Ca2+ peaks and slowed Ca2+ removal, but not by increased markers of oxidative stress or impaired mitochondrial respiration. No relevant increases in muscle carnosine content were observed after carnosine supplementation. Results show that a primary function of HCDs in cardiac muscle is the regulation of Ca2+ handling and excitation-contraction coupling.

Passive skeletal muscle can function as an osmotic engine.

Muscles are composite structures. The protein filaments responsible for force production are bundled within fluid-filled cells, and these cells are wrapped in ordered sleeves of fibrous collagen. Recent models suggest that the mechanical interaction between the intracellular fluid and extracellular collagen is essential to force production in passive skeletal muscle, allowing the material stiffness of extracellular collagen to contribute to passive muscle force at physiologically relevant muscle lengths. Such models lead to the prediction, tested here, that expansion of the fluid compartment within muscles should drive forceful muscle shortening, resulting in the production of mechanical work unassociated with contractile activity. We tested this prediction by experimentally increasing the fluid volumes of isolated bullfrog semimembranosus muscles via osmotically hypotonic bathing solutions. Over time, passive muscles bathed in hypotonic solution widened by 16.44 ± 3.66% (mean ± s.d.) as they took on fluid. Concurrently, muscles shortened by 2.13 ± 0.75% along their line of action, displacing a force-regulated servomotor and doing measurable mechanical work. This behaviour contradicts the expectation for an isotropic biological tissue that would lengthen when internally pressurized, suggesting a functional mechanism analogous to that of engineered pneumatic actuators and highlighting the significance of three-dimensional force transmission in skeletal muscle.

Effect of acute nitrite infusion on contractile economy and metabolism in isolated skeletal muscle in situ during hypoxia.

Increased plasma nitrite concentrations may have beneficial effects on skeletal muscle function. The physiological basis explaining these observations has not been clearly defined and it may involve positive effects on muscle contraction force, microvascular O2 delivery and skeletal muscle oxidative metabolism. In the isolated canine gastrocnemius model, we evaluated the effects of acute nitrite infusion on muscle force and skeletal muscle oxidative metabolism. Under hypoxic conditions, but in the presence of normal convective O2 delivery, an elevated plasma nitrite concentration affects neither muscle force, nor muscle contractile economy. In accordance with previous results suggesting limited or no effects of nitrate/nitrite administrations in highly oxidative and highly perfused muscle, our data suggest that neither mitochondrial respiration, nor muscle force generation are affected by acute increased concentrations of NO precursors in hypoxia. Contrasting findings have been reported concerning the effects of augmented nitric oxide (NO) on skeletal muscle force production and oxygen consumption ( ). The present study examined skeletal muscle mitochondrial respiration and contractile economy in an isolated muscle preparation during hypoxia (but normal convective O2 delivery) with nitrite infusion. Isolated canine gastrocnemius muscles in situ (n=8) were studied during 3min of electrically stimulated isometric tetanic contractions corresponding to ∼35% of . During contractions, sodium nitrite (NITRITE) or sodium chloride (SALINE) was infused into the popliteal artery. was calculated from the Fick principle. Experiments were carried out in hypoxia ( =0.12), whereas convective O2 delivery was maintained at normal levels under both conditions by pump-driven blood flow ( ). Muscle biopsies were taken and mitochondrial respiration was evaluated by respirometry. Nitrite infusion significantly increased both nitrite and nitrate concentrations in plasma. No differences in force were observed between conditions. was not significantly different between NITRITE (6.1±1.8mL 100g-1 min-1 ) and SALINE (6.2±1.8mL100g-1 min-1 ), even after being 'normalized' per unit of developed force (muscle contractile economy). No differences between conditions were found for maximal ADP-stimulated mitochondrial respiration (both for complex I and complex II), leak respiration and oxidative phosphorylation coupling. In conclusion, in the absence of changes in convective O2 delivery, muscle force, muscle contractile economy and mitochondrial respiration were not affected by acute infusion of nitrite. The previously reported positive effects of elevated plasma nitrite concentrations are presumably mediated by the increased microvascular O2 availability.

Skeletal Muscle Mitochondrial Adaptations to Maximal Strength Training in Older Adults.

Maximal strength training (MST) results in robust improvements in skeletal muscle force production, efficiency, and mass. However, the effects of MST on muscle mitochondria are still unknown. Accordingly, the purpose of this study was to examine, from the molecular level to whole-muscle, mitochondrial adaptations induced by 8 weeks of knee-extension MST in the quadriceps of 10 older adults using immunoblotting, spectrophotometry, high-resolution respirometry in permeabilized muscle fibers, in vivo 31P magnetic resonance spectroscopy (31P-MRS), and gas exchange. As anticipated, MST resulted in an increased isometric knee-extensor force from 133 ± 36 to 147 ± 49 Nm (p < .05) and quadriceps muscle volume from 1,410 ± 103 to 1,555 ± 455 cm3 (p < .05). Mitochondrial complex (I-V) protein abundance and citrate synthase activity were not significantly altered by MST. Assessed ex vivo, maximal ADP-stimulated respiration (state 3CI+CII, PRE: 23 ± 6 and POST: 14 ± 5 ρM·mg-1·s-1, p < .05), was decreased by MST, predominantly, as a result of a decline in complex I-linked respiration (p < .05). Additionally, state 3 free-fatty acid linked respiration was decreased following MST (PRE: 19 ± 5 and POST: 14 ± 3 ρM·mg-1·s-1, p < .05). Assessed in vivo, MST slowed the PCr recovery time constant (PRE: 49 ± 13 and POST: 57 ± 16 seconds, p < .05) and lowered, by ~20% (p = .055), the quadriceps peak rate of oxidative ATP synthesis, but did not significantly alter the oxidation of lipid. Although these, likely qualitative, mitochondrial adaptations are potentially negative in terms of skeletal muscle energetic capacity, they need to be considered in light of the many improvements in muscle function that MST affords older adults.

Incubation with sodium nitrite attenuates fatigue development in intact single mouse fibres at physiological .

Dietary nitrate supplementation increases plasma nitrite concentration, which provides an oxygen-independent source of nitric oxide and can delay skeletal muscle fatigue. Nitrate supplementation has been shown to increase myofibre calcium release and force production in mouse skeletal muscle during contractions at a supra-physiological oxygen tension, but it is unclear whether nitrite exposure can delay fatigue development and improve myofibre calcium handling at a near-physiological oxygen tension. Single mouse muscle fibres acutely treated with nitrite had a lower force and cytosolic calcium concentration during single non-fatiguing contractions at a near-physiological oxygen tension. Nitrite treatment delayed fatigue development during repeated fatiguing isometric contractions at near-physiological, but not at supra-physiological, oxygen tension in combination with better maintenance of myofilament calcium sensitivity and sarcoplasmic reticulum calcium pumping. These findings improve understanding of the mechanisms by which increased skeletal muscle nitrite exposure might be ergogenic and imply that this is related to improved calcium handling. Dietary nitrate (NO3- ) supplementation, which increases plasma nitrite (NO2- ) concentration, has been reported to attenuate skeletal muscle fatigue development. Sarcoplasmic reticulum (SR) calcium (Ca2+ ) release is enhanced in isolated single skeletal muscle fibres following NO3- supplementation or NO2- incubation at a supra-physiological but it is unclear whether NO2- incubation can alter Ca2+ handling and fatigue development at a near-physiological . We hypothesised that NO2- treatment would improve Ca2+ handling and delay fatigue at a physiological in intact single mouse skeletal muscle fibres. Each muscle fibre was perfused with Tyrode solution pre-equilibrated with either 20% ( ∼150Torr) or 2% O2 ( =15.6Torr) in the absence and presence of 100µM NaNO2 . At supra-physiological (i.e. 20% O2 ), time to fatigue was lowered by 34% with NaNO2 (control: 257±94 vs. NaNO2 : 159±46s, Cohen's d=1.63, P<0.05), but extended by 21% with NaNO2 at 2% O2 (control: 308±217 vs. NaNO2 : 368±242s, d=1.14, P<0.01). During the fatiguing contraction protocol completed with NaNO2 at 2% O2 , peak cytosolic Ca2+ concentration ([Ca2+ ]c ) was not different (P>0.05) but [Ca2+ ]c accumulation between contractions was lower, concomitant with a greater SR Ca2+ pumping rate (P<0.05) compared to the control condition. These results demonstrate that increased exposure to NO2- blunts fatigue development at near-physiological, but not at supra-physiological, through enhancing SR Ca2+ pumping rate in single skeletal muscle fibres. These findings extend our understanding of the mechanisms by which increased NO2- exposure can mitigate skeletal muscle fatigue development.