Effects of sclerostin injection on soleus and extensor digitorum longus muscle tissue in male mice.

Sclerostin, a potent inhibitor of the Wnt signaling pathway, plays a critical role in bone homeostasis. Evidence suggests that sclerostin may also be involved in crosstalk between other tissues, including muscle. This pilot study attempted to examine the effects of sclerostin on soleus and extensor digitorum longus (EDL) muscle tissue from male mice that were given continuous recombinant sclerostin injections for 4 weeks. A total of 48 10-week-old male C57BL/6J mice were assigned to be sedentary or perform 1 h treadmill running per day for 4 weeks and administered subcutaneous injections of either saline or recombinant sclerostin 5 days/week. Sclerostin injection led to a reduction in the soleus myosin heavy chain (MHC) I, MHC I/IIA, MHC IIA/X, and MHC IIB cross-sectional area (p < 0.05) with no exercise effects on these reductions. In contrast, there were no effects of sclerostin injections or exercise on the fast-twitch EDL muscle in terms of size, MHC protein, or markers of Wnt signaling. These findings provide preliminary evidence of sclerostin's endocrine role in muscle via decreases in myofiber cross-sectional area, which seems to be independent of fiber type but muscle type-specific. More studies, however, are needed to confirm these preliminary results.

Clinical practice guideline for management of osteoporosis and fracture prevention in Canada: 2023 update.

BACKGROUND: In Canada, more than 2 million people live with osteoporosis, a disease that increases the risk for fractures, which result in excess mortality and morbidity, decreased quality of life and loss of autonomy. This guideline update is intended to assist Canadian health care professionals in the delivery of care to optimize skeletal health and prevent fractures in postmenopausal females and in males aged 50 years and older. METHODS: This guideline is an update of the 2010 Osteoporosis Canada clinical practice guideline on the diagnosis and management of osteoporosis in Canada. We followed the Grading of Recommendations Assessment, Development and Evaluation (GRADE) framework and quality assurance as per Appraisal of Guidelines for Research and Evaluation (AGREE II) quality and reporting standards. Primary care physicians and patient partners were represented at all levels of the guideline committees and groups, and participated throughout the entire process to ensure relevance to target users. The process for managing competing interests was developed before and continued throughout the guideline development, informed by the Guideline International Network principles. We considered benefits and harms, patient values and preferences, resources, equity, acceptability and feasibility when developing recommendations; the strength of each recommendation was assigned according to the GRADE framework. RECOMMENDATIONS: The 25 recommendations and 10 good practice statements are grouped under the sections of exercise, nutrition, fracture risk assessment and treatment initiation, pharmacologic interventions, duration and sequence of therapy, and monitoring. The management of osteoporosis should be guided by the patient's risk of fracture, based on clinical assessment and using a validated fracture risk assessment tool. Exercise, nutrition and pharmacotherapy are key elements of the management strategy for fracture prevention and should be individualized. INTERPRETATION: The aim of this guideline is to empower health care professionals and patients to have meaningful discussions on the importance of skeletal health and fracture risk throughout older adulthood. Identification and appropriate management of skeletal fragility can reduce fractures, and preserve mobility, autonomy and quality of life.

Potentiation of force by extracellular potassium and posttetanic potentiation are additive in mouse fast-twitch muscle in vitro.

The influence of moderately elevated extracellular potassium concentration ([K+]) on muscle force has marked similarities to that of posttetanic potentiation (PTP) in that twitch force may be enhanced whilst high-frequency force is depressed. The purpose of this work was to test whether K+-induced potentiation is mechanistically related to PTP via skeletal myosin light-chain kinase (skMLCK)-catalyzed phosphorylation of the myosin regulatory light chains (RLC). To do this, we assessed the influence of elevated [K+] on the force response at various frequencies in extensor digitorum longus (EDL) muscles isolated from wild-type and skeletal myosin light-chain kinase (skMLCK-/-) absent mice. Changing [K+] of the incubation medium from 5 to 10 mmol increased isometric twitch force by a similar amount in wild-type and skMLCK-/- muscles (~ 13% in both genotypes) (all data n = 7-8, P < 0.05). In contrast, 100- and 200-Hz forces were depressed in both genotypes (by 5-7 and 15-18%, respectively). The isometric twitch potentiation caused by a tetanic stimulus series was similar at both [K+] levels for each genotype but was much greater for wild-type than for skMLCK-/- muscles (i.e., 23-25 and 8-9%, respectively). Thus, we conclude that [K+]- and stimulation-induced potentiation are additive and that [K+]-induced potentiation is independent of RLC phosphorylation.

Myosin phosphorylation improves contractile economy of mouse fast skeletal muscle during staircase potentiation.

Phosphorylation of the myosin regulatory light chain (RLC) by skeletal myosin light chain kinase (skMLCK) potentiates rodent fast twitch muscle but is an ATP-requiring process. Our objective was to investigate the effect of skMLCK-catalyzed RLC phosphorylation on the energetic cost of contraction and the contractile economy (ratio of mechanical output to metabolic input) of mouse fast twitch muscle in vitro (25°C). To this end, extensor digitorum longus (EDL) muscles from wild-type (WT) and from skMLCK-devoid (skMLCK-/-) mice were subjected to repetitive low-frequency stimulation (10 Hz for 15 s) to produce staircase potentiation of isometric twitch force, after which muscles were quick frozen for determination of high-energy phosphate consumption (HEPC). During stimulation, WT muscles displayed significant potentiation of isometric twitch force while skMLCK-/- muscles did not (i.e. 23% versus 5% change, respectively). Consistent with this, RLC phosphorylation was increased ∼3.5-fold from the unstimulated control value in WT but not in skMLCK-/- muscles. Despite these differences, the HEPC of WT muscles was not greater than that of skMLCK-/- muscles. As a result of the increased contractile output relative to HEPC, the calculated contractile economy of WT muscles was greater than that of skMLCK-/- muscles. Thus, our results suggest that skMLCK-catalyzed phosphorylation of the myosin RLC increases the contractile economy of WT mouse EDL muscle compared with skMLCK-/- muscles without RLC phosphorylation.

Myosin phosphorylation potentiates steady-state work output without altering contractile economy of mouse fast skeletal muscles.

Skeletal myosin light chain kinase (skMLCK)-catalyzed phosphorylation of the myosin regulatory light chain (RLC) increases (i.e. potentiates) mechanical work output of fast skeletal muscle. The influence of this event on contractile economy (i.e. energy cost/work performed) remains controversial, however. Our purpose was to quantify contractile economy of potentiated extensor digitorum longus (EDL) muscles from mouse skeletal muscles with (wild-type, WT) and without (skMLCK ablated, skMLCK-/-) the ability to phosphorylate the RLC. Contractile economy was calculated as the ratio of total work performed to high-energy phosphate consumption (HEPC) during a period of repeated isovelocity contractions that followed a potentiating stimulus (PS). Consistent with genotype, the PS increased RLC phosphorylation measured during, before and after isovelocity contractions in WT but not in skMLCK-/- muscles (i.e. 0.65 and 0.05 mol phosphate mol-1 RLC, respectively). In addition, although the PS enhanced work during repeated isovelocity contractions in both genotypes, the increase was significantly greater in WT than in skMLCK-/- muscles (1.51±0.03 versus 1.10±0.05, respectively; all data P<0.05, n=8). Interestingly, the HEPC determined during repeated isovelocity contractions was statistically similar between genotypes at 19.03±3.37 and 16.02±3.41 µmol P; respectively (P<0.27). As a result, despite performing significantly more work, the contractile economy calculated for WT muscles was similar to that calculated for skMLCK-/- muscles (i.e. 5.74±0.67 and 4.61±0.71 J kg-1 µmol-1 P, respectively (P<0.27). In conclusion, our results support the notion that myosin RLC phosphorylation enhances dynamic contractile function of mouse fast skeletal muscle but does so without decreasing contractile economy.

Actualisation 2023 des lignes directrices de pratique clinique pour la prise en charge de l'ostéoporose et la prévention des fractures au Canada.

CONTEXTE: Au Canada, plus de 2 millions de personnes vivent avec l'ostéoporose, une maladie qui accroît le risque de fracture, ce qui fait augmenter la morbidité et la mortalité, et entraîne une perte de qualité de vie et d'autonomie. La présente actualisation des lignes directrices vise à accompagner les professionnelles et professionnels de la santé au Canada dans la prestation de soins visant à optimiser la santé osseuse et à prévenir les fractures chez les femmes ménopausées et les hommes de 50 ans et plus. MÉTHODES: Le présent document fournit une actualisation des lignes directrices de pratique clinique de 2010 d'Ostéoporose Canada sur le diagnostic et la prise en charge de l'ostéoporose au pays. Nous avons utilisé l'approche GRADE (Grading of Recommendations Assessment, Development and Evaluation) et effectué l'assurance de la qualité conformément aux normes de qualité et de présentation des rapports de la grille AGREE II (Appraisal of Guidelines for Research & Evaluation). Les médecins de premier recours et les patientes et patients partenaires ont été représentés à tous les niveaux des comités et des groupes ayant participé à l'élaboration des lignes directrices, et ont participé à toutes les étapes du processus pour garantir la pertinence des informations pour les futurs utilisateurs et utilisatrices. Le processus de gestion des intérêts concurrents a été entamé avant l'élaboration des lignes directrices et s'est poursuivi sur toute sa durée, selon les principes du Réseau international en matière de lignes directrices. Dans la formulation des recommandations, nous avons tenu compte des avantages et des risques, des valeurs et préférences de la patientèle, des ressources, de l'équité, de l'acceptabilité et de la faisabilité; la force de chacune des recommandations a été déterminée en fonction du cadre GRADE. RECOMMANDATIONS: Les 25 recommandations et les 10 énoncés de bonne pratique sont répartis en sections : activité physique, alimentation, évaluation du risque de fracture, instauration du traitement, interventions pharmacologiques, durée et séquence du traitement, et monitorage. La prise en charge de l'ostéoporose devrait se fonder sur le risque de fracture, établi au moyen d'une évaluation clinique réalisée avec un outil d'évaluation du risque de fracture validé. L'activité physique, l'alimentation et la pharmacothérapie sont des éléments essentiels à la stratégie de prévention des fractures, qui devraient être personnalisés. INTERPRÉTATION: Les présentes lignes directrices ont pour but d'outiller les professionnelles et professionnels de la santé et la patientèle afin qu'ensemble ils puissent parler de l'importance de la santé osseuse et du risque de fracture tout au long de la vie adulte avancée. La détection et la prise en charge efficace de la fragilité osseuse peuvent contribuer à réduire les fractures et à préserver la mobilité, l'autonomie et la qualité de vie.

Shortening speed dependent force potentiation is attenuated but not eliminated in skeletal muscles without myosin phosphorylation.

We investigated the influence of shortening speed on concentric force potentiation at different frequencies in muscles devoid of skeletal myosin light chain kinase (skMLCK-/-) and unable to phosphorylate myosin. EDL muscles from skMLCK-/- mice were activated in vitro (25 °C) across a range of stimulation frequencies (10-100 Hz) during shortening ramps at 0.10, 0.30, or 0.50 of maximum shortening velocity (Vmax) before and after a potentiating stimulus (PS). When collapsed across all frequencies, the PS increased relative (post/pre) concentric force to 1.27 ± 0.02 and 1.17 ± 0.02 of pre-PS values at 0.50 and 0.30 Vmax, respectively (n = 4, P < 0.05 for all speeds). In addition, potentiation was significantly greater at low and intermediate-than at high stimulus frequencies at both speeds. In contrast, during shortening at 0.10 Vmax, a posttetanic depression was observed as mean concentric forces were reduced to 0.85 ± 0.02 of pre-PS values. Thus, although reduced compared to published values for wildtype muscles (Gittings et al., J Muscle Res Cell Motil 33:359-368, 2012), skMLCK-/- muscles displayed a speed dependent potentiation of concentric force during moderate and fast shortening speed at all frequencies tested. Our data support the presence of a myosin phosphorylation-independent mechanism(s) for concentric force potentiation at moderate speeds of shortening, and also suggests that myosin phosphorylation may be necessary to prevent the concentric force depression that may be present at slow speeds of shortening. Although additive in nature, further work is needed to parse out the relative influence of myosin phosphorylation-independent and dependent potentiation mechanisms on wildtype contractile function during dynamic conditions.

Myosin light chain phosphorylation is required for peak power output of mouse fast skeletal muscle in vitro.

The skeletal myosin light chain kinase (skMLCK) catalyzed phosphorylation of the myosin regulatory light chain (RLC) is associated with potentiation of force, work, and power in rodent fast twitch muscle. The purpose of this study was to compare concentric responses of EDL from wild-type (WT) and skMLCK devoid (skMLCK-/-) muscles at a range of shortening speeds (0.05 to 0.70 V max) around that expected to produce maximal power (in vitro, 25 °C) both before (unpotentiated) and after (potentiated) a potentiating stimulus (PS). When collapsed across all speeds tested, neither unpotentiated force, work, or power differed between genotypes (all data n = 10, P < 0.05). In contrast, although both genotypes displayed speed-dependent increases, these increases were greater for WT than skMLCK-/- muscles. For example, when collapsed across the six fastest speeds we tested, both concentric force and power were increased 30-34 % in WT but only 15-17 % in skMLCK-/- muscles. In contrast, at the two slowest speeds, these parameters were increased in WT but decreased in skMLCK-/- muscles (8-10 and 7-9 %, respectively). Intriguingly, potentiation of concentric force and power was optimal near speeds producing maximal power in both genotypes. Because the PS elevated RLC phosphorylation above resting levels in WT but not in skMLCK-/- muscles, our data suggest that skMLCK-catalyzed phosphorylation of the RLC is required for maximal concentric power output of mouse EDL muscle stimulated at high frequency in vitro.

Interaction of posttetanic potentiation and the catchlike property in mouse skeletal muscle.

INTRODUCTION: Posttetanic potentiation (PTP) and the catchlike property (CLP) enhance contractile function in skeletal muscle. We investigated the CLP during dynamic performance in mouse hindlimb muscles with (wild-type) and without (skMLCK(-/-) ) the primary mechanism for PTP (myosin phosphorylation) (in vitro, 25°C). METHODS: Extensor digitorum longus muscles of both genotypes were stimulated with constant frequency and catchlike trains (CFT and CLT), before and after a potentiating stimulus (PS). RESULTS: Before the PS, the CLT increased concentric force/work relative to the CFT, but this effect was greater for skMLCK(-/-) than wild-type muscles. After the PS, the catchlike effect was reduced in wild-type muscles but unchanged in skMLCK(-/-) muscles that did not display PTP. CONCLUSIONS: These data suggest that PTP interferes with the CLP during concentric force development at moderate speeds of shortening. We conclude that the physiological utility of each mechanism and their interactions provide important modulations to fast skeletal muscle function. Muscle Nerve 54: 308-316, 2016.

The force dependence of isometric and concentric potentiation in mouse muscle with and without skeletal myosin light chain kinase.

The isometric potentiation associated with myosin phosphorylation is force dependent. The purpose of this study was to assess the influence of a pre-existing period of isometric force on the concentric force potentiation displayed by mouse muscles with and without the ability to phosphorylate myosin. We tested isometric (ISO) and concentric (CON) potentiation, as well as concentric potentiation after isometric force (ISO-CON), in muscles from wild-type (WT) and skeletal myosin light chain kinase-deficient (skMLCK(-/-)) mice. A conditioning stimulus increased (i.e., potentiated) mean concentric force in the ISO-CON and CON conditions to 1.31 ± 0.02 and 1.35 ± 0.02 (WT) and to 1.19 ± 0.02 and 1.21 ± 0.01 (skMLCK(-/-)) of prestimulus levels, respectively (data n = 6-8, p < 0.05). No potentiation of mean isometric force was observed in either genotype. The potentiation of mean concentric force was inversely related to relative tetanic force level (P/Po) in both genotypes. Moreover, concentric potentiation varied greatly within each contraction type and was negatively correlated with unpotentiated force in both genotypes. Thus, although no effect of pre-existing force was observed, strong and inverse relationships between concentric force potentiation and unpotentiated concentric force may suggest an influence of attached and force-generating crossbridges on potentiation magnitude in both WT and skMLCK(-/-) muscles.

Maternal folic acid supplementation does not impact skeletal muscle function and metabolism in male and female CD-1 mouse offspring.

Folic acid fortification of all white flour, enriched pasta, and cornmeal products became mandatory in Canada to reduce risk of neural tube defects at birth. Furthermore, Health Canada and the Society of Obstetricians and Gynaecologists of Canada recommend women take daily prenatal folic acid supplements in addition to folic acid fortified foods during pregnancy. However, the influence of maternal folic acid supplementation on offspring development, specifically the highly abundant and metabolically active skeletal muscle, is currently unknown. Thus, the purpose of this study was to determine the effect of supplemental folic acid (four times higher than normal dietary consumption), in utero and throughout suckling on muscle size, function, and metabolism in male and female CD-1 mouse offspring. The major findings were that maternal exposure to supplemental folic acid (i) had no impact on postpartum growth rates or muscle mass in female and male offspring, (ii) had no impact on skeletal muscle contractile kinetics in females and male offspring, and (iii) increased maximal phosphofructokinase activity in extensor digitorum longus of female and male offspring. These findings suggest that exposure to folic acid supplementation in utero and throughout suckling at levels four times higher than recommended had minimal effect on skeletal muscle size, function, and metabolism regardless of sex. Future research is needed explore the underlying biological pathways and mechanisms affected by folic acid supplementation during pregnancy and lactation on offspring skeletal muscle tissue, specifically in humans.

Myosin phosphorylation and force potentiation in skeletal muscle: evidence from animal models.

The contractile performance of mammalian fast twitch skeletal muscle is history dependent. The effect of previous or ongoing contractile activity to potentiate force, i.e. increase isometric twitch force, is a fundamental property of fast skeletal muscle. The precise manifestation of force potentiation is dependent upon a variety of factors with two general types being identified; staircase potentiation referring to the progressive increase in isometric twitch force observed during low frequency stimulation while posttetanic potentiation refers to the step-like increase in isometric twitch force observed following a brief higher frequency (i.e. tetanic) stimulation. Classic studies established that the magnitude and duration of potentiation depends on a number of factors including muscle fiber type, species, temperature, sarcomere length and stimulation paradigm. In addition to isometric twitch force, more recent work has shown that potentiation also influences dynamic (i.e. concentric and/or isotonic) force, work and power at a range of stimulus frequencies in situ or in vitro, an effect that may translate to enhanced physiological function in vivo. Early studies performed on both intact and permeabilized models established that the primary mechanism for this modulation of performance was phosphorylation of myosin, a modification that increased the Ca(2+) sensitivity of contraction. More recent work from a variety of muscle models indicates, however, the presence of a secondary mechanism for potentiation that may involve altered Ca(2+) handling. The primary purpose of this review is to highlight these recent findings relative to the physiological utility of force potentiation in vivo.

Sclerostin Influences Exercise-Induced Adaptations in Body Composition and White Adipose Tissue Morphology in Male Mice.

Sclerostin is an inhibitor of the osteogenic Wnt/ß-catenin signaling pathway that also has an endocrine role in regulating adipocyte differentiation and metabolism. Additionally, subcutaneous white adipose tissue (scWAT) sclerostin content decreases following exercise training (EXT). Therefore, we hypothesized that EXT-induced reductions in adipose tissue sclerostin may play a role in regulating adaptations in body composition and whole-body metabolism. To test this hypothesis, 10-week-old male C57BL/6J mice were either sedentary (SED) or performing 1 hour of treadmill running at ~65% to 70% maximum oxygen consumption (VO2max ) 5 day/week (EXT) for 4 weeks and had subcutaneous injections of either saline (C) or recombinant sclerostin (S) (0.1 mg/kg body mass) 5 day/week; thus, making four groups (SED-C, EXT-C, SED-S, and EXT-S; n = 12/group). No differences in body mass were observed between experimental groups, whereas food intake was higher in EXT (p = 0.03) and S (p = 0.08) groups. There was a higher resting energy expenditure in all groups compared to SED-C. EXT-C had increased lean mass and decreased fat mass percentage compared to SED-C and SED-S. No differences in body composition were observed in either the SED-S or EXT-S groups. Lower scWAT (inguinal), epididymal white adipose tissue (eWAT) (visceral epididymal) mass, and scWAT adipocyte cell size and increased percentage of multilocular cells in scWAT were observed in the EXT-C group compared to SED-C, whereas lower eWAT was only observed in the EXT-S group. EXT mice had increased scWAT low-density lipoprotein receptor-related protein 4 (Lrp4) and mitochondrial content and sclerostin treatment only inhibited increased Lrp4 content with EXT. Together, these results provide evidence that reductions in resting sclerostin with exercise training may influence associated alterations in energy metabolism and body composition, particularly in scWAT. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Toward countering muscle and bone loss with spaceflight: GSK3 as a potential target.

We examined the effects of ∼30 days of spaceflight on glycogen synthase kinase 3 (GSK3) content and inhibitory serine phosphorylation in murine muscle and bone samples from four separate missions (BION-M1, rodent research [RR]1, RR9, and RR18). Spaceflight reduced GSK3ß content across all missions, whereas its serine phosphorylation was elevated with RR18 and BION-M1. The reduction in GSK3ß was linked to the reduction in type IIA fibers commonly observed with spaceflight as these fibers are particularly enriched with GSK3. We then tested the effects of inhibiting GSK3 before this fiber type shift, and we demonstrate that muscle-specific Gsk3 knockdown increased muscle mass, preserved muscle strength, and promoted the oxidative fiber type with Earth-based hindlimb unloading. In bone, GSK3 activation was enhanced after spaceflight; and strikingly, muscle-specific Gsk3 deletion increased bone mineral density in response to hindlimb unloading. Thus, future studies should test the effects of GSK3 inhibition during spaceflight.

Tetanic force potentiation of mouse fast muscle is shortening speed dependent.

The activity dependent potentiation of peak isometric force associated with phosphorylation of the myosin regulatory light chain (RLC) is generally restricted to low activation frequencies. The purpose of this study was to determine if muscle shortening speed influenced the stimulus frequency domain over which concentric force potentiation was observed. To this end, mouse extensor digitorum longus (EDL) muscles (in vitro, 25 °C) were activated at a range of test frequencies (10, 25, 45, 70 or 100 Hz) during shortening ramps at 0.10, 0.30 or 0.50 of the maximal velocity of shortening (V(max)). This procedure was performed before and after a standard conditioning stimulus (CS) that elevated RLC phosphorylation from 0.08 ± 0.01 (rest) to 0.55 ± 0.01 (stimulated) moles phosphate per mol RLC, respectively (n = 9-11) (P < 0.01). When data from all test frequencies were collapsed, the CS potentiated mean concentric force at 0.10, 0.30 and 0.50 V(max) to 1.02 ± 0.03, 1.37 ± 0.03 and 1.59 ± 0.05 of unpotentiated, pre-CS values, respectively (n = 8, P < 0.05). In addition, increasing shortening speed also increased the activation frequency at which concentric force potentiation was maximal, i.e. from 10 Hz at 0.10 V(max) to 10-25 and 25-45 Hz at 0.30 and 0.50 V(max), respectively. These results indicate that both the magnitude of and activation frequency dependence for concentric force potentiation of mouse EDL muscle is shortening speed dependent. Thus, muscle shortening speed may be a critical factor determining the functional utility of the myosin RLC phosphorylation mechanism.

Myosin light-chain phosphorylation and potentiation of dynamic function in mouse fast muscle.

The intent of this study was to determine if the stimulation-induced increase or "potentiation" of dynamic function of mouse extensor digitorum longus muscle (in vitro 25°C) during work cycles is graded to myosin regulatory light-chain (RLC) phosphorylation. To do this, concentric force and muscle work output during sinusoidal length changes were determined before (unpotentiated) and after (potentiated) the application of conditioning stimuli (CS) producing incremental elevations in RLC phosphorylation from rest. Sine wave excursion was from 1.09 to 0.91 of L (o) with a period of 142 ms; stimulating muscles to twitch and generate force during these cycles produced plots of force × displacement termed work loops. Stimulation at 2.5-, 5.0-, and 100-Hz elevated RLC phosphorylation from 0.16±0.02 (rest) to 0.29±0.03, 0.45±0.02 and 0.56±0.02 mol phos per mole RLC, respectively (n= 6-7, P<0.05). These CS potentiated mean concentric force (at all lengths) to 1.14±0.02, 1.26±0.04 and 1.41±0.06 of pre-stimulus, control levels (all n= 5-7, P<0.05) while work was increased to 1.07±0.02, 1.17±0.02 and 1.34±0.03 of controls, respectively. In a No CS condition that did not elevate RLC phosphorylation, neither mean concentric force nor work was altered. Thus, strong correlations between RLC phosphorylation and mean concentric force and work support the hypothesis that this molecular mechanism modulates muscle power output. No length-dependence for concentric force potentiation was observed in any condition, an outcome suggesting that interactions between instantaneous variations in muscle length and shortening velocity during work cycles modulates the potentiation response.

PDH activation during in vitro muscle contractions in PDH kinase 2 knockout mice: effect of PDH kinase 1 compensation.

Pyruvate dehydrogenase (PDH) plays an important role in regulating carbohydrate oxidation in skeletal muscle. PDH is deactivated by a set of PDH kinases (PDK1, PDK2, PDK3, PDK4), with PDK2 and PDK4 being the most predominant isoforms in skeletal muscle. Although PDK2 is the most abundant isoform, few studies have examined its physiological role. The role of PDK2 on PDH activation (PDHa) at rest and during muscle stimulation at 10 and 40 Hz (eliciting low- and moderate-intensity muscle contractions, respectively) in isolated extensor digitorum longus muscles was studied in PDK2 knockout (PDK2KO) and wild-type (WT) mice (n = 5 per group). PDHa activity was unexpectedly 35 and 77% lower in PDK2KO than WT muscle (P = 0.043), while total PDK activity was nearly fourfold lower in PDK2KO muscle (P = 0.006). During 40-Hz contractions, initial force was lower in PDK2KO than WT muscle (P < 0.001) but fatigued similarly to ∼75% of initial force by 3 min. There were no differences in initial force or rate of fatigue during 10-Hz contractions. PDK1 compensated for the lack of PDK2 and was 1.8-fold higher in PDK2KO than WT muscle (P = 0.019). This likely contributed to ensuring that resting PDHa activity was similar between the groups and accounts for the lower PDH activation during muscle contraction, as PDK1 is a very potent inhibitor of the PDH complex. Increased PDK1 expression appears to be regulated by hypoxia inducible factor-1α, which was 3.5-fold higher in PDK2KO muscle. It is clear that PDK2 activity is essential, even at rest, in regulation of carbohydrate oxidation and production of reducing equivalents for the electron transport chain. In addition, these results underscore the importance of the overall kinetics of the PDK isoform population, rather than total PDK activity, in determining transformation of the PDH complex and PDHa activity during muscle contraction.

The effect of skeletal myosin light chain kinase gene ablation on the fatigability of mouse fast muscle.

Contraction-induced activation of a skeletal muscle specific Ca(2+) and calmodulin dependent myosin light chain kinase (skMLCK) catalyzes phosphorylation of the myosin regulatory light chain (RLC), a reaction that potentiates twitch force. The purpose of this study was to test the effect of skMLCK gene ablation on the fatigability of mouse extensor digitorum longus (EDL) muscle (in vitro at 25°C). Muscles were isolated from wildtype (WT, n = 10-12) and skeletal MLCK knockout (skMLCK KO, n = 10-12) mice and fatigued using a protocol consisting of 5 min of repeated tetanic stimulation (150 Hz for 1000 ms every 5 s). Both twitch (P(t)) and tetanic (P(o)) force as well as unloaded shortening velocity (V(o)) were assessed before, during and after fatiguing stimulation. Fatiguing stimulation increased RLC phosphorylation in WT but not skMLCK KO muscles (16 ± 0.01-0.63 ± 0.02 and 0.07 ± 0.02-0.08 ± 0.02 mol phos mol RLC, respectively). Although P(t) was potentiated above baseline in both WT and KO muscles, this increase was greater in WT than in KO muscles (to 1.37 ± 0.05 vs. 1.14 ± 0.02 of unpotentiated values, respectively). The difference in P(t) persisted until P(o) had been diminished to ~60% of baseline and thereafter P(t) declined to similar levels in both WT and KO muscles (to ~35% of initial). Overall, the time-course and decline in P(o) for WT and KO was similar (reduced to 0.20 ± 0.01 and 0.20 ± 0.01 of baseline, respectively) (P < 0.05). Initial values for V(o) were similar between WT and KO muscles and, moreover, the fatigue related decline in Vo was similar for both muscle genotypes (P < 0.05). Thus, our results demonstrate that skMLCK--catalyzed RLC phosphorylation augments isometric twitch force during moderate, but not severe, levels of fatigue.

The effect of work cycle frequency on the potentiation of dynamic force in mouse fast twitch skeletal muscle.

The purpose of this study was to test the hypothesis that the potentiation of concentric twitch force during work cycles is dependent upon both the speed and direction of length change. Concentric and eccentric forces were elicited by stimulating muscles during the shortening and lengthening phases, respectively, of work cycles. Work cycle frequency was varied in order to vary the speed of muscle shortening and/or lengthening; all forces were measured as the muscle passed though optimal length (L(o)). Both concentric and eccentric force were assessed before (unpotentiated control) and after (potentiated) the application of a tetanic conditioning protocol known to potentiate twitch force output. The influence of the conditioning protocol on relative concentric force was speed dependent, with forces increased to 1.19±0.01, 1.25±0.01 and 1.30±0.01 of controls at 1.5, 3.3 and 6.9 Hz, respectively (all data N=9-10 with P<0.05). In contrast, the conditioning protocol had only a limited effect on eccentric force at these frequencies (range: 1.06±0.01 to 0.96±0.03). The effect of the conditioning protocol on concentric work (force × distance) was also speed dependent, being decreased at 1.5 Hz (0.84±0.01) and increased at 3.3 and 6.9 Hz (1.05±0.01 and 1.39±0.01, respectively). In contrast, eccentric work was not increased at any frequency (range: 0.88±0.02 to 0.99±0.01). Thus, our results reveal a hysteresis-like influence of activity-dependent potentiation such that concentric force and/or work were increased but eccentric force and/or work were not. These outcomes may have implications for skeletal muscle locomotor function in vivo.

Black and Green Tea as Well as Specialty Teas Increase Osteoblast Mineralization with Varying Effectiveness.

Many human studies suggest a benefit of tea consumption on bone health. The study objective was to compare the ability of different tea types to promote mineralization. Saos-2 cells underwent mineralization (5 days) in the presence of tea (white: WT, green: GT, black: BT, green rooibos: GR, or red rooibos: RR; 1 µg/mL of polyphenols) or control. Total polyphenol content (TPC, Folin-Ciocalteu's reagent), antioxidant capacity (2,2-diphenyl-1-picrylhydrazyl [DPPH] scavenging), mineralization (Alizarin Red staining), gene expression quantitative reverse transcription PCR (RT-qPCR), and cell activity (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay) were determined. TPC was highest in GT and BT. The ability of each tea to inhibit DPPH also differed (WT, GT > RR) after normalizing for polyphenol quantity. Each tea increased mineralization and differences were observed among types (GT/BT/GR/RR > WT, GT = BT = GR, RR > BT/GT). mRNA expression of alkaline phosphatase (ALP) and ectonucleotide pyrophosphatase/phosphodiesterase (NPP1) remained unchanged, whereas osteopontin (OPN) and sclerostin (SOST) were reduced in cells treated with tea, regardless of type. At 24- and 48-h postexposure to tea, cell activity was greater in cells receiving any of the teas compared with vehicle control. Supplementation increased mineralization regardless of tea type with both rooibos teas and black tea stimulating greater mineralization than WT, whereas green tea is similar to the others. While future study is needed to confirm in vivo effects, the results suggest that consuming any of the teas studied may benefit bone health.