Human Vastus Lateralis Muscle Research Articles

Fatiguing exercise reduces cellular passive Young's modulus in human vastus lateralis muscle.

Previous studies demonstrated that acute fatiguing exercise transiently reduces whole-muscle stiffness, which might contribute to increased risk of injury and impaired contractile performance. We sought to elucidate potential intracellular mechanisms underlying these reductions. To that end, the cellular passive Young's modulus was measured in muscle fibres from healthy, young males and females. Eight volunteers (four male and four female) completed unilateral, repeated maximal voluntary knee extensions until task failure, immediately followed by bilateral percutaneous needle muscle biopsy of the post-fatigued followed by the non-fatigued control vastus lateralis. Muscle samples were processed for mechanical assessment and separately for imaging and phosphoproteomics. Fibres were passively (pCa 8.0) stretched incrementally to 156% of initial sarcomere length to assess Young's modulus, calculated as the slope of the resulting stress-strain curve at short (sarcomere length=2.4-3.0µm) and long (sarcomere length=3.2-3.8µm) lengths. Titin phosphorylation was assessed by liquid chromatography followed by high-resolution mass spectrometry. The passive modulus was significantly reduced in post-fatigued versus control fibres from male, but not female, participants. Post-fatigued samples showed altered phosphorylation of five serine residues (four located within the elastic region of titin) but did not exhibit altered active tension or sarcomere ultrastructure. Collectively, these results suggest that acute fatigue is sufficient to alter phosphorylation of skeletal titin in multiple locations. We also found reductions in the passive modulus, consistent with prior reports in the literature investigating striated muscle stiffness. These results provide mechanistic insight contributing to the understanding of dynamic regulation of whole-muscle tissue mechanics in vivo. HIGHLIGHTS: What is the central question of this study? Previous studies have shown that skeletal muscle stiffness is reduced following a single bout of fatiguing exercise in whole muscle, but it is not known whether these changes manifest at the cellular level, and their potential mechanisms remain unexplored. What is the main finding and its importance? Fatiguing exercise reducescellular stiffnessin skeletal muscle from males but not females, suggesting thatfatigue alters tissue compliance in asex-dependent manner. The phosphorylation status of titin, a potential mediator of skeletal muscle cellular stiffness, is modified by fatiguing exercise. Previous studies have shown that passive skeletal muscle stiffness is reduced following a single bout of fatiguing exercise. Lower muscle passive stiffness following fatiguing exercise might increase risk for soft-tissue injury; however, the underlying mechanisms of this change are unclear. Our findings show that fatiguing exercise reduces the passive Young's modulus in skeletal muscle cells from males but not females, suggesting that intracellular proteins contribute to reduced muscle stiffness following repeated loading to task failure in a sex-dependent manner. The phosphorylation status of the intracellular protein titin is modified by fatiguing exercise in a way that might contribute to altered muscle stiffness after fatiguing exercise. These results provide important mechanistic insight that might help to explain why biological sex impacts the risk for soft-tissue injury with repeated or high-intensity mechanical loading in athletes and the risk of falls in older adults.

GDF5 as a rejuvenating treatment for age-related neuromuscular failure.

Sarcopenia involves a progressive loss of skeletal muscle force, quality and mass during ageing, which results in increased inability and death; however, no cure has been established thus far. Growth differentiation factor 5 (GDF5) has been described to modulate muscle mass maintenance in various contexts. For our proof of concept, we overexpressed GDF5 by AAV vector injection in Tibialis Anterior (TA) muscle of adult aged (20 months) mice and performed molecular and functional analysis of skeletal muscle. We analysed human Vastus Lateralis muscle biopsies from adult young (21-42 years) and aged (77-80 years) donors, quantifying the molecular markers modified by GDF5 overexpression (OE) in mouse muscle. We validated the major effects of GDF5 overexpression using human immortalized myotubes and Schwann Cells (SCs). We established a pre-clinical study by treating chronically (for 4 months) aged mice using recombinant GDF5 protein (rGDF5) in systemic administration and evaluated the long-term effect of this treatment on muscle mass and function. Here, we demonstrated that GDF5 OE in the old TAs promoted an increase of 16.5% of muscle weight (P = 0.0471) associated with a higher percentage of 5000-6000 µm2 large fibres (P = 0.0211), without the induction of muscle regeneration. Muscle mass gain was associated with an amelioration of 26.8% of rate of force generation (P = 0.0330) and a better neuromuscular connectivity (P = 0.0098). Moreover, GDF5 OE preserved neuromuscular junction (NMJ) morphology (38.5% of nerve terminal area increase, P < 0.0001) and stimulated the expression of re-innervation-related genes, in particular markers of SCs (fold change 3.19 for S100b gene expression, P = 0.0101). To further characterize the molecular events induced by GDF5 OE during ageing, we performed a genome-wide transcriptomic analysis of treated muscles and showed that this factor leads to a "rejuvenating" transcriptomic signature in aged mice, as 42% of the transcripts dysregulated by ageing reverted to youthful expression levels upon GDF5 OE (P < 0.05). Towards a pre-clinical approach, we performed a long-term systemic treatment using rGDF5 and showed its effectiveness in counteracting age-related muscle wasting, improving muscle function (17,8% of absolute maximal force increase, P = 0.0079), ensuring neuromuscular connectivity and preventing NMJ degeneration (7,96% of AchR area increase, P = 0.0125). In addition, in human muscle biopsies, we found the same age-related alterations than those observed in mice and improved by GDF5 and reproduced its major effects on human cells, suggesting this treatment as efficient in humans. Overall, these data provide a foundation to examine the curative potential of GDF5 drug in clinical trials for sarcopenia and, eventually, other neuromuscular diseases.

Fascicle shortening upon activation in voluntary human muscle contractions

BACKGROUND: The dependence of fascicle length on complex interactions with joint angle and force challenges the interpretation of in vivo joint mechanics, muscle mechanical properties, contractile behavior, and muscle function. AIM: The purpose of this study was to determine the complex interaction between muscle activation, joint angle, and fascicle length for isometric contractions of the human vastus lateralis muscle (VL). METHOD: Knee extensor torques, joint angles, EMG activation, and fascicle lengths were determined in nine healthy subjects during maximal and submaximal isometric contractions. RESULTS: Fascicle shortening during isometric contractions depended on muscle-tendon unit length/joint angle and activation, reaching a maximum between angles where VL had its maximum force potential and minimum resistance to fascicle shortening. Maximal fascicle shortening shifted to shorter muscle-tendon unit lengths with decreasing activation. CONCLUSION: Fascicle shortening upon activation depends crucially on the force generating potential and stiffness of the muscle and can reach 30% of the resting fascicle length. Not accounting for the complex interactions between muscle length, force potential, muscle structure, and muscle stiffness has led to erroneous interpretations of the function and properties of healthy and diseased muscles.

4. Skeletal Muscle in Inbred Mice Exhibits Divergent Hypoxia and Angiogenesis Transcriptome Expression as Compared to Humans in Aging

Hypoxia signaling has been previously demonstrated to be a critical regulator in age-dependent skeletal muscle regeneration. While animal models provide a crucial scientific substrate for medical innovation and translation, discordant findings have been commonly described in existing literature between animals and humans. The nature and extent of this divergence in the context of aging and its effect on skeletal muscle regeneration remains incompletely understood. Quadriceps muscles from young (Y, 12 weeks, n=3) and old (O, 25 months, n=3) C57BL/6 mice were harvested and analyzed via RNA bulk-sequencing. These results were compared to healthy human vastus lateralis muscles (Y, 22-30 years, n=8; O, 80-83 years, n=5) from the GESTALT study (NIA/NIH) analyses as found in the Gene Expression Omnibus (NIH) database. Gross examination of the most differentially expressed genes demonstrated low variance between samples within each mouse cohort (Disp = 0.04467, BCV = 0.2114) relative to young/old human (Disp = 0.61261, BCV = 0.7827) specimens. Enrichment analyses were agnostically performed on filtered differentially expressed human genes via edgeR (Bioconductor) resulting in significant overlap with the HIF-1 signaling pathway KEGG term (Eno1, Tfrc, Ldha, Eno3, and Pdha1) with significant upregulation in young human muscles. However, these same genes demonstrated no significant differences within mice. Interestingly, when examining angiogenesis gene lists determined a priori, mice demonstrated several significantly upregulated genes in the young cohort (Anxa2, Lox, Mmp9, Angptl4, Adora2b, and Pgf) that thematically correspond to modulators of the extracellular matrix in the process of neoangiogenesis. Conversely, none of these genes were found to be significantly different between old and young humans. To further compare the muscle transcriptomes between humans and mice in the context of aging, genes with the largest magnitude fold changes and most significant p-values were identified. The most significant differentially expressed genes in mice demonstrated robust upregulation of Mss51, Igkv3-2, Ighg2c, Rasd2, Igkc, Eda2r, and Gdf11 in old mice. These same genes in human specimens demonstrated no comparable segregation. Similarly, the most upregulated genes in old human specimens (Gda, Mtcybp32, Fgf7, Fam83b, Lyve1, Plag1, Eda2r, Dkk2, and Col21a1) showed no similar segregations in mice. The most differentially expressed genes for hypoxia and angiogenesis in both mice and humans were not found to be concordantly upregulated when cross-examined across the other species. Thus, skeletal muscle in inbred mice exhibits differential transcriptome expression and decreased variance as compared to humans in aging. Given these findings, great care should be taken in making inferences between findings between mice and humans. As NextGen sequencing becomes routine, incorporation of genetically diverse mice to better approximate human genetic variation may provide further insights in translation from bench to bedside.

Effect of eccentric and concentric contraction mode on myogenic regulatory factors expression in human vastus lateralis muscle.

Skeletal muscle contractions are caused to release myokines by muscle fiber. This study investigated the myogenic regulatory factors, as MHC I, IIA, IIX, Myo-D, MRF4, Murf, Atrogin-1, Decorin, Myonection, and IL-15 mRNA expression in the response of eccentric vs concentric contraction. Eighteen healthy men were randomly divided into two eccentric and concentric groups, each of 9 persons. Isokinetic contraction protocols included maximal single-leg eccentric or concentric knee extension tasks at 60°/s with the dominant leg. Contractions consisted of a maximum of 12 sets of 10 reps, and the rest time between each set was 30s. The baseline biopsy was performed 4 weeks before the study, and post-test biopsies were taken immediately after exercise protocols from the vastus lateralis muscle. The gene expression levels were evaluated using Real-Time PCR methods. The eccentric group showed a significantly lower RPE score than the concentric group (P ≤ 0.05). A significant difference in MyoD, MRF4, Myonection, and Decorin mRNA, were observed following eccentric or concentric contractions (P ≤ 0.05). The MHC I, MHC IIA, IL-15 mRNA has been changed significantly compared to the pre-exercise in the concentric group (P ≤ 0.05). While only MHC IIX and Atrogin-1 mRNA changed significantly in the eccentric group (P ≤ 0.05). Additionally, the results showed a significant difference in MyoD, MRF4, IL-15, and Decorin at the follow-up values between eccentric or concentric groups (P ≤ 0.05). Our findings highlight the growing importance of elucidating the different responses of muscle growth factors associated with a myogenic activity such as MHC IIA, Decorin, IL-15, Myonectin, Decorin, MuRF1, and MHC IIX mRNA in following various types of exercise.

Muscle Fiber Types in Human Vastus Lateralis muscle by Oxidative Stains

Muscle Fiber Types in Human Vastus Lateralis muscle by Oxidative Stains

Negligible epimuscular myofascial force transmission between the human rectus femoris and vastus lateralis muscles in passive conditions.

There have been contradictory reports of the effects of epimuscular myofascial force transmission in humans. This study investigated the transmission of myofascial force to the human vastus lateralis muscle by determining whether vastus lateralis slack angle changed with hip angle. Since the distance between the origin and insertion of the vastus lateralis muscle does not change when hip angle changes, any change in vastus lateralis slack angle with hip position can be attributed to epimuscular myofascial force transmission. Nineteen young adults were tested in hip flexed ([Formula: see text]) and neutral ([Formula: see text]) positions. Ultrasound images of the vastus lateralis muscle were obtained as the knee was passively flexed at [Formula: see text]/s. The knee angle at which vastus lateralis muscle fascicles began to lengthen was used to identify muscle slack angle. Overall, there was a negligible effect of hip position on vastus lateralis slack angle ([Formula: see text] [[Formula: see text] to 1.9]; mean [95% confidence interval]). However, a small and variable effect was noted in 3/19 participants. This result indicates that, over the range of joint angles tested here, there is little or no epimuscular myofascial force transmission between the vastus lateralis muscle and neighbouring bi-articular structures under passive conditions. More broadly, this result provides additional evidence that epimuscular myofascial force transmission tends to be small and variable under passive conditions in healthy human muscle.

Rates of oxidative ATP synthesis are not augmented beyond the pH threshold in human vastus lateralis muscles during a stepwise contraction protocol.

The oxygen cost of high-intensity exercise at power outputs above an individual's lactate threshold (LT) is greater than would be predicted by the linear oxygen consumption-power relationship observed below the LT. However, whether these augmentations are caused by an increased ATP cost of force generation (ATPCOST ) or an increased oxygen cost of ATP synthesis is unclear. We used 31 P-MRS to measure changes in cytosolic [ADP] (intramyocellular marker of oxidative metabolism), oxidative ATP synthesis (ATPOX ) and ATPCOST during a 6-stage, stepwise knee extension protocol. ATPCOST was unchanged across stages. The relationship between [ADP] and muscle power output was augmented at workloads above the pH threshold (pHT ; proxy for LT), whereas increases in ATPOX were attenuated. These results suggest the greater oxygen cost of contractions at workloads beyond the pHT is not caused by mechanisms that increase ATPCOST , but rather mechanisms that alter intrinsic mitochondrial function or capacity. Increases in skeletal muscle metabolism and oxygen consumption are linearly related to muscle power output for workloads below the lactate threshold (LT), but are augmented (i.e. greater rate of increase relative to workload) thereafter. Presently, it is unclear whether these metabolic augmentations are caused by increases in the ATP cost of force generation (ATPCOST ) or changes in the efficiency of mitochondrial oxygen consumption and oxidative ATP synthesis (ATPOX ). To partition these two hypotheses in vivo, we used 31 P-MRS to calculate slopes relating step-changes in muscle work to concurrent changes in cytosolic phosphates and ATPOX before and after the pH threshold (pHT ; used here as a proxy for LT) within the vastus lateralis muscle of eight young adults during a stepwise knee extension test. Changes in muscle phosphates and ATPOX were linearly related to workload below the pHT . However, slopes above the pHT were greater for muscle phosphates (P<0.05) and lower for ATPOX (P<0.05) than were the slopes observed below the pHT . The maximal capacity for ATPOX ( ) and ADP-specific ATPOX also declined beyond the pHT (P<0.05), whereas ATPCOST was unchanged (P=0.10). These results oppose the hypothesis that high-intensity contractions increase ATPCOST and suggest that greater oxidative metabolism at workloads beyond the pHT is caused by mechanisms that affect intrinsic mitochondrial function or capacity, such as alterations in substrate selection or electron entry into the electron transport chain, temperature-mediated changes in mitochondrial permeability to protons, or stimulation of mitochondrial uncoupling by reactive oxygen species generation.

Paxilline Prevents the Onset of Myotonic Stiffness in Pharmacologically Induced Myotonia: A Preclinical Investigation

Reduced Cl− conductance causes inhibited muscle relaxation after forceful voluntary contraction due to muscle membrane hyperexcitability. This represents the pathomechanism of myotonia congenita. Due to the prevailing data suggesting that an increased potassium level is a main contributor, we studied the effect of a modulator of a big conductance Ca2+- and voltage-activated K+ channels (BK) modulator on contraction and relaxation of slow- and high-twitch muscle specimen before and after the pharmacological induction of myotonia. Human and murine muscle specimens (wild-type and BK−/−) were exposed to anthracene-9-carboxylic acid (9-AC) to inhibit CLC-1 chloride channels and to induce myotonia in-vitro. Functional effects of BK-channel activation and blockade were investigated by exposing slow-twitch (soleus) and fast-twitch (extensor digitorum longus) murine muscle specimens or human musculus vastus lateralis to an activator (NS1608) and a blocker (Paxilline), respectively. Muscle-twitch force and relaxation times (T90/10) were monitored. Compared to wild type, fast-twitch muscle specimen of BK−/− mice resulted in a significantly decreased T90/10 in presence of 9-AC. Paxilline significantly shortened T90/10 of murine slow- and fast-twitch muscles as well as human vastus lateralis muscle. Moreover, twitch force was significantly reduced after application of Paxilline in myotonic muscle. NS1608 had opposite effects to Paxilline and aggravated the onset of myotonic activity by prolongation of T90/10. The currently used standard therapy for myotonia is, in some individuals, not very effective. This in vitro study demonstrated that a BK channel blocker lowers myotonic stiffness and thus highlights its potential therapeutic option in myotonia congenital (MC).

Effect of different muscle contraction mode on the expression of Myostatin, IGF-1, and PGC-1 alpha family members in human Vastus Lateralis muscle.

Muscle contraction stimulates a transient change of myogenic factors, partly related to the mode of contractions. Here, we assessed the response of IGF-1Ea, IGF-1Eb, IGF-1Ec, PGC1α-1, PGC1α-4, and myostatin to the eccentric Vs. the concentric contraction in human skeletal muscle. Ten healthy males were performed an acute eccentric and concentric exercise bout (n = 5 per group). For each contraction type, participants performed 12 sets of 10 repetitions knee extension by the dominant leg. Baseline and post-exercise muscle biopsy were taken 4weeks before and immediately after experimental sessions from Vastus Lateralis muscle. Genes expression was measured by real-time PCR technique. There was a significant increase in PGC1α-1, PGC1α-4, IGF-1Ea and, IGF-1Eb mRNA after concentric contraction (p ≤ 0.05), while the PGC1α-4 and IGF-1Ec significantly increased after eccentric contraction (p ≤ 0.05). It is intriguing to highlight that; no significant differences between groups were evident for changes in any variables following exercise bouts (p ≥ 0.05). Our results found that concentric and eccentric contractions presented different responses in PGC1α-1, IGF-1Ea, IGF-1Eb, and IGF-1Ec mRNA. However, a similar significant increase in mRNA content was observed in PGC1α-4. Further, no apparent differences could be found between the response of genes to eccentric and concentric contraction.

History-dependence of muscle slack length in humans: effects of contraction intensity, stretch amplitude, and time.

The slack length of a relaxed skeletal muscle can be reduced by isometric contraction at short lengths ("contract-short conditioning"). This study explored how the effect of contract-short conditioning on muscle slack length is modified by 1) the intensity of the contraction, 2) the delay between the contraction and measurement of slack length, and 3) the amplitude of a stretch delivered to the relaxed muscle after the contraction. Muscle fascicles in the human vastus lateralis muscle were observed with ultrasound imaging while the relaxed muscle was lengthened by flexing the knee. The knee angle at which muscle fascicle slack was taken up was used as a proxy for muscle slack length. Conditioning the muscle with voluntary isometric (fixed-end) contractions at short muscle lengths reduced vastus lateralis muscle slack length, measured 60 s later, by a mean of 10°. This effect was independent of contraction intensity from 5% to 100% maximal voluntary contraction. The effect was largest when first observed 5 s after the contraction, decayed about one-third by 60 s, and then remained nearly constant until the last observation 5 min after the contraction. A slow stretch given to the relaxed muscle after contract-short conditioning increased slack length (i.e., reduced the effect of contract-short conditioning). Slack length increased nonlinearly with stretch amplitude. Very large stretches (>30°, possibly as large as 90°) were required to abolish the effect of contract-short conditioning. The phenomena described here share some characteristics with, and may involve similar mechanisms to, passive force enhancement and muscle thixotropy.NEW & NOTEWORTHY The slack length of a relaxed human skeletal muscle is not fixed; it can be modified by contraction and stretch. Contraction of the human vastus lateralis muscle at short lengths reduces the muscle's slack length. Even very weak contractions are sufficient to induce this effect. The effect persists for at least 5 min but can be reduced or abolished with a large-amplitude passive stretch.

Human skeletal muscle fiber type percentage and area after reduced muscle use: A systematic review and meta-analysis.

The main objective of this systematic review was to examine the effect of reduced muscle activity on the relative number of type 1 muscle fibers (%) in the human vastus lateralis muscle. Other objectives were changes in type 2A and 2X percentages and muscle fiber cross-sectional area. We conducted systematic literature searches in eight databases and included studies assessing type 1 fiber percentage visualized by ATPase or immunohistochemical staining before and after a period (≥14days) of reduced muscle activity. The reduced muscle activity models were detraining, leg unloading, and bed rest. Forty-two studies comprising 451 participants were included. Effect sizes were calculated as the mean difference between baseline and follow-up and Generic Inverse Variance tests with random-effects models were used for the weighted summary effect size. Overall, the mean type 1 muscle fiber percentage was significantly reduced after interventions (-1.94%-points, 95% CI [-3.37, -0.51], P=.008), with no significant differences between intervention models (P=.86). Meta-regression showed no effect of study duration on type 1 fiber percentage (P=.98). Conversely, the overall type 2X fiber percentage increased after reduced muscle activity (P<.001). The CSA of the muscle fiber types decreased after the study period (all P-values<0.001) with greater reductions in type 2 than type 1 fibers (P<.001). The result of this meta-analysis display that the type 1 muscle fiber percentage decrease as a result of reduced muscle activity, although the effect size is relatively small.

A Mathematical Model Linking Ca2+-Dependent Signaling Pathway and Gene Expression Regulation in Human Skeletal Muscle

The physiological adaptation to aerobic endurance exercises is provided by activation of signaling pathways in skeletal muscle cells. Training-induced activation of specific signaling pathways results in significant transcriptional responses. Despite the ongoing endeavours to experimentally investigate regulatory mechanisms and signal transduction pathways involved in the contraction-induced adaptation, quantitative contribution of certain signal molecules in expression regulation of genes responsible for intracellular response has not been studied comprehensively yet. The paper presents novel developed model linking Ca2+-dependent signaling pathway and downstream transcription regulation of early and late response genes in human skeletal muscle during exercise. Numerical analysis of the model enabled to reveal crucial steps in this signal transduction pathway for the adaptation and demonstrated the necessity of consideration of additional transcription factors regulating transcription of late response genes in order to adequately reproduce gene expression data that were taken in human vastus lateralis muscle during and after acute cycling exercise.

Skeletal muscle unloading results in increased mitophagy and decreased mitochondrial biogenesis regulation

IntroductionPhysical inactivity significantly contributes to loss of muscle mass and performance in bed‐bound patients. Loss of skeletal muscle mitochondrial content has been well‐established in muscle unloading models, but the underlying molecular mechanism remains unclear. We hypothesized that apparent unloading‐induced loss of muscle mitochondrial content is preceded by increased mitophagy‐ and decreased mitochondrial biogenesis‐signaling during the early stages of unloading.MethodsWe analyzed a comprehensive set of molecular markers involved in mitochondrial‐autophagy, −biogenesis, −dynamics, and ‐content, in the gastrocnemius muscle of C57BL/6J mice subjected to 0‐ and 3‐days hind limb suspension, and in biopsies from human vastus lateralis muscle obtained before and after 7 days of one‐leg immobilization.ResultsIn both mice and men, short‐term skeletal muscle unloading results in molecular marker patterns indicative of increased receptor‐mediated mitophagy and decreased mitochondrial biogenesis regulation, before apparent loss of mitochondrial content.DiscussionThese results emphasize the early‐onset of skeletal muscle disuse‐induced mitochondrial remodeling.

1779-P: Characterization of Insulin-Regulated CLASP2 Phosphorylation

During an unbiased proteomics screen of rat L6 myotubes, we previously discovered the first member of the microtubule plus-end interacting protein (+TIP) family that undergoes insulin-stimulated phosphorylation, CLIP-associating protein 2 (CLASP2). Here we report that CLASP2 protein is expressed in several muscle groups as well as epididymal fat in mouse and we have successfully confirmed the expression of CLASP2 protein in human vastus lateralis muscle by mass spectrometry. In addition, insulin resistant, 60% fat diet-fed mice exhibit a loss of CLASP2 expression in epididymal adipose tissue versus control animals. We performed an insulin time course and discovered that insulin stimulates the phosphorylation of both endogenous and overexpressed CLASP2 in 3T3-L1 adipocytes in a manner to that similarly observed in L6 myotubes. Inhibition of either PI 3-kinase with wortmannin or mTOR with rapamycin had no effect on insulin-stimulated, proline-directed serine phosphorylation of CLASP2, whereas inhibition of the MAPK pathway reduced proline-directed serine phosphorylation of endogenous CLASP2 to basal levels. Quantitative phosphoproteomics identified Ser884/Ser1004/Ser1005/Ser1021 as significant insulin-stimulated CLASP2 phosphorylation sites that can be decreased after inhibition of the MAPK pathway. These findings indicate that CLASP2 undergoes insulin-stimulated proline-directed serine phosphorylation that is under control of the MAPK pathway in 3T3-L1 adipocytes. In addition, we also present new evidence of CLASP2 protein expression in mammalian tissues that are specifically targeted for insulin-stimulated glucose uptake and suggest that CLASP2 is altered within in vivo models of insulin resistance and type 2 diabetes. Disclosure N.K. Barker: None. J.L. Krantz: None. P.R. Langlais: None. Funding National Institutes of Health

Neurovascular structures in human vastus lateralis muscle and the ideal biopsy site.

A density model of neurovascular structures was generated from 28 human vastus lateralis muscles isolated from embalmed cadavers. The intramuscular portion of arteries, veins, and nerves was dissected, traced on transparencies, and digitized before adjustment to an average muscle shape using Procrustes analysis to generate density distributions for the relative positions of these structures. The course of arteries, veins, and nerves was highly variable between individual muscles. Nevertheless, a zone of lower average neurovascular density was found between the tributaries from the lateral circumflex femoral and the deep femoral arteries. While the area with the lowest density was covered by the iliotibial tract and would therefore not be suitable for biopsies, another low-density area was located in the distal portion of vastus lateralis. This was just anterior to the iliotibial tract, in a zone that has been described as a good needle biopsy site. The reported complication rates of needle biopsies (0.1%-4%) are in the range of expectations when simulated based on this model. It is concluded that the optimal human vastus lateralis biopsy site is in the distal portion of the muscle, between ½ and ¾ of the length from the greater trochanter to the lateral epicondyle, just anterior to the iliotibial band.

History-dependence of muscle slack length following contraction and stretch in the human vastus lateralis.

In reduced muscle preparations, the slack length and passive stiffness of muscle fibres have been shown to be influenced by previous muscle contraction or stretch. In human muscles, such behaviours have been inferred from measures of muscle force, joint stiffness and reflex magnitudes and latencies. Using ultrasound imaging, we directly observed that isometric contraction of the vastus lateralis muscle at short lengths reduces the slack lengths of the muscle-tendon unit and muscle fascicles. The effect is apparent 60s after the contraction. These observations imply that muscle contraction at short lengths causes the formation of bonds which reduce the effective length of structures that generate passive tension in muscles. In reduced muscle preparations, stretch and muscle contraction change the properties of relaxed muscle fibres. In humans, effects of stretch and contraction on properties of relaxed muscles have been inferred from measurements of time taken to develop force, joint stiffness and reflex latencies. The current study used ultrasound imaging to directly observe the effects of stretch and contraction on muscle-tendon slack length and fascicle slack length of the human vastus lateralis muscle in vivo. The muscle was conditioned by (a) strong isometric contractions at long muscle-tendon lengths, (b) strong isometric contractions at short muscle-tendon lengths, (c) weak isometric contractions at long muscle-tendon lengths and (d) slow stretches. One minute after conditioning, ultrasound images were acquired from the relaxed muscle as it was slowly lengthened through its physiological range. The ultrasound image sequences were used to identify muscle-tendon slack angles and fascicle slack lengths. Contraction at short muscle-tendon lengths caused a mean 13.5degree (95% CI 11.8-15.0degree) shift in the muscle-tendon slack angle towards shorter muscle-tendon lengths, and a mean 5mm (95% CI 2-8mm) reduction in fascicle slack length, compared to the other conditions. A supplementary experiment showed the effect could be demonstrated if the muscle was conditioned by contraction at short lengths but not if the relaxed muscle was held at short lengths, confirming the role of muscle contraction. These observations imply that muscle contraction at short lengths causes the formation of bonds which reduce the effective length of structures that generate passive tension in muscles.

Dominant and sensitive control of oxidative flux by the ATP-ADP carrier in human skeletal muscle mitochondria: Effect of lysine acetylation

The adenine nucleotide translocase (ANT) of the mitochondrial inner membrane exchanges ADP for ATP. Mitochondria were isolated from human vastus lateralis muscle (n = 9). Carboxyatractyloside titration of O2 consumption rate (Jo) at clamped [ADP] of 21 μM gave ANT abundance of 0.97 ± 0.14 nmol ANT/mg and a flux control coefficient of 82% ± 6%. Flux control fell to 1% ± 1% at saturating (2 mM) [ADP]. The KmADP for Jo was 32.4 ± 1.8 μM. In terms of the free (−3) ADP anion this KmADP was 12.0 ± 0.7 μM. A novel luciferase-based assay for ATP production gave KmADP of 13.1 ± 1.9 μM in the absence of ATP competition. The free anion KmADP in this case was 2.0 ± 0.3 μM. Targeted proteomic analyses showed significant acetylation of ANT Lysine23 and that ANT1 was the most abundant isoform. Acetylation of Lysine23 correlated positively with KmADP, r = 0.74, P = 0.022. The findings underscore the central role played by ANT in the control of oxidative phosphorylation, particularly at the energy phosphate levels associated with low ATP demand. As predicted by molecular dynamic modeling, ANT Lysine23 acetylation decreased the apparent affinity of ADP for ANT binding.

Operating length and velocity of human vastus lateralis muscle during walking and running

According to the force-length-velocity relationships, the muscle force potential during locomotion is determined by the operating fibre length and velocity. We measured fascicle and muscle-tendon unit length and velocity as well as the activity of the human vastus lateralis muscle (VL) during walking and running. Furthermore, we determined the VL force-length relationship experimentally and calculated the force-length and force-velocity potentials (i.e. fraction of maximum force according to the force-length-velocity curves) for both gaits. During the active state of the stance phase, fascicles showed significantly (p < 0.05) smaller length changes (walking: 9.2 ± 4.7% of optimal length (L0); running: 9.0 ± 8.4%L0) and lower velocities (0.46 ± 0.36 L0/s; 0.03 ± 0.83 L0/s) compared to the muscle-tendon unit (walking: 19.7 ± 5.3%L0, −0.94 ± 0.32 L0/s; running: 34.5 ± 5.8%L0, −2.59 ± 0.41 L0/s). The VL fascicles operated close to optimum length (L0 = 9.4 ± 0.11 cm) in both walking (8.6 ± 0.14 cm) and running (10.1 ± 0.19 cm), resulting in high force-length (walking: 0.92 ± 0.08; running: 0.91 ± 0.14) and force-velocity (0.91 ± 0.08; 0.97 ± 0.13) potentials. For the first time we demonstrated that, in contrast to the current general conception, the VL fascicles operate almost isometrically and close to L0 during the active state of the stance phase of walking and running. The findings further verify an important contribution of the series-elastic element to VL fascicle dynamics.

Protein translation, proteolysis and autophagy in human skeletal muscle atrophy after spinal cord injury.

Spinal cord injury-induced loss of skeletal muscle mass does not progress linearly. In humans, peak muscle loss occurs during the first 6 weeks postinjury, and gradually continues thereafter. The aim of this study was to delineate the regulatory events underlying skeletal muscle atrophy during the first year following spinal cord injury. Key translational, autophagic and proteolytic proteins were analysed by immunoblotting of human vastus lateralis muscle obtained 1, 3 and 12 months following spinal cord injury. Age-matched able-bodied control subjects were also studied. Several downstream targets of Akt signalling decreased after spinal cord injury in skeletal muscle, without changes in resting Akt Ser473 and Akt Thr308 phosphorylation or total Akt protein. Abundance of mTOR protein and mTOR Ser2448 phosphorylation, as well as FOXO1 Ser256 phosphorylation and FOXO3 protein, decreased in response to spinal cord injury, coincident with attenuated protein abundance of E3 ubiquitin ligases, MuRF1 and MAFbx. S6 protein and Ser235/236 phosphorylation, as well as 4E-BP1 Thr37/46 phosphorylation, increased transiently after spinal cord injury, indicating higher levels of protein translation early after injury. Protein abundance of LC3-I and LC3-II decreased 3 months postinjury as compared with 1 month postinjury, but not compared to able-bodied control subjects, indicating lower levels of autophagy. Proteins regulating proteasomal degradation were stably increased in response to spinal cord injury. Together, these data provide indirect evidence suggesting that protein translation and autophagy transiently increase, while whole proteolysis remains stably higher in skeletal muscle within the first year after spinal cord injury.