N pi-methylhistidine in myosin-light-chain kinase

The identification and localization of N pi-methylhistidine (1-methylhistidine) within the primary structure (His 157) of rabbit skeletal muscle myosin-light-chain kinase is described. The data show for the first time that N pi-methylhistidine, known as a rare mammalian urine amino acid, is a protein constituent.     

Force-velocity relationships in hypertensive arterial smooth muscle

Increased total peripheral resistance is the cardinal haemodynamic disorder in essential hypertension. This could be secondary to alterations in the mechanical properties of vascular smooth muscle. Adequate study has not been made of the force-velocity (F-V) relationship in hypertensive arterial smooth muscle. Increased shortening in arterial smooth muscle would result in greater narrowing of arteries. The objectives of this investigation were to see if there is (i) increased shortening or increased maximum change in muscle length (delta Lmax where L stands for muscle length), (ii) an increased maximum velocity of shortening (Vmax) measured in l omicron per second where l omicron is the optimal muscle length for tension development, and (iii) a difference in maximum isometric tension (P omicron) developed in spontaneously hypertensive rat (SHR; N = 6) compared with normotensive Wistar Kyoto rat (WKY;N = 5) caudal artery strips. An electromagnetic muscle lever was employed in recording force-velocity data. Analysis of these data revealed the following: (a) the SHR mean P omicron of 6.21 +/- 1.01 N/cm2 was not different from the mean WKY P omicron of 6.97 +/- 1.64 N/cm2 (p greater than 0.05); (b) the SHR preparations showed greater shortening for all loads imposed; (c) the SHR Vmax of 0.016 l omicron/s was greater than the WKY Vmax of 0.013 l omicron/s (p less than 0.05). This study provides evidence that while hypertensive arterial smooth muscle is not able to produce more force than normotensive arterial smooth muscle, it is capable of faster and greater shortening. The latter could result in increased narrowing of hypertensive arteries and increased blood pressure.     

Foot and ankle forces during an automobile collision: the influence of muscles

Muscles have a potentially important effect on lower extremity injuries during an automobile collision. Computational modeling can be a powerful tool to predict these effects and develop protective interventions. Our purpose was to determine how muscles influence peak foot and ankle forces during an automobile collision. A 2-D bilateral musculoskeletal model was constructed with seven segments. Six muscle groups were included in the right lower extremity, each represented by a Hill muscle model. Vehicle deceleration data were applied as input and the resulting movements were simulated. Three models were evaluated: no muscles (NM), minimal muscle activation at a brake pedal force of 400 N (MN), and maximal muscle activation to simulate panic braking (MX). Muscle activation always resulted in large increases in peak joint force. Peak ankle joint force was greatest for MX (10120 N), yet this model also had the lowest peak rearfoot force (629 N). Peak force on the Achilles tendon was 4.5 times greater, during MX (6446 N) compared to MN (1430 N). We conclude that (1). external and internal forces are dependent on muscles, (2). muscle activation level could exacerbate axial loading injuries, (3). external and internal forces can be inversely related once muscle properties are included.     

Measurement of human Gracilis muscle isometric forces as a function of knee angle,intraoperatively

The goals of the present study were (1) to measure the previously unstudied isometric forces of activated human Gracilis (G) muscle as a function of knee joint angle and (2) to test whether length history effects are important also for human muscle. Experiments were conducted intraoperatively during anterior cruciate ligament (ACL) reconstruction surgery (n=8). Mean peak G muscle force, mean peak G tendon stress and mean optimal knee angle equals 178.5±270.3 N, 24.4±20.6 MPa and 67.5±41.7°, respectively. The substantial inter-subject variability found (e.g., peak G force ranges between 17.2 and 490.5 N) indicate that the contribution of the G muscle to knee flexion moment may vary considerably among subjects. Moreover, typical subject anthropometrics did not appear to provide a sound estimate of the peak G force: only a limited insignificant correlation was found between peak G force and subject mass as well as mid-thigh perimeter and no correlation was found between peak G force and thigh length. The functional joint range of motion for human G muscle was determined to be at least as wide as full knee extension to 120° of knee flexion. However; the portion of the knee angle–muscle force relationship operationalized is not unique but individual specific: our data suggest for most subjects that G muscle operates in both ascending and descending limbs of its length–force characteristics whereas, for the remainder of the subjects, its function is limited to the descending limb, exclusively. Previous activity of G muscle at high muscle length attained during collection of a complete set of knee angle–force data showed for the first time that such length history effects are important also for human muscles: a significant correlation was found between optimal knee angle and absolute value of % force change. Except for two of the subjects, G muscle force measured at low length was lower than that measured during collection of knee joint–force data (maximally by 42.3%).     

Reappraisal of the quantitative importance of non-skeletal-muscle source of N tau-methylhistidine in urine.

The claim of Millward, Bates, Grimble, Brown, Nathan & Rennie [(1980) Biochem. J. 190. 225--228] that muscle actomyosin contributes as little as 25% of urinary N tau-methylhistidine is not consistent with other published data from that group [Bates, DeCoster, Grimble, Holloszy, Millward & Rennie (1980) J. Physiol. (London) 303, 41 P] or with literature values. It appears likely that the turnover rate of muscle actomyosin has been considerably underestimated and that when realistic rates of protein turnover are used, muscle tissue remains the major contributor of N tau-methylhistidine in urine.     

A comparison of a maximum exertion method and a model-based, sub-maximum exertion method for normalizing trunk EMG

The problem with normalizing EMG data from patients with painful symptoms (e.g., low back pain) is that such patients may be unwilling or unable to perform maximum exertions. Furthermore, the normalization to a reference signal, obtained from a maximal or sub-maximal task, tends to mask differences that might exist as a result of pathology. Therefore, we presented a novel method (GAIN method) for normalizing trunk EMG data that overcomes both problems. The GAIN method does not require maximal exertions (MVC) and tends to preserve distinct features in the muscle recruitment patterns for various tasks. Ten healthy subjects performed various isometric trunk exertions, while EMG data from 10 muscles were recorded and later normalized using the GAIN and MVC methods. The MVC method resulted in smaller variation between subjects when tasks were executed at the three relative force levels (10%, 20%, and 30% MVC), while the GAIN method resulted in smaller variation between subjects when the tasks were executed at the three absolute force levels (50 N, 100 N, and 145 N). This outcome implies that the MVC method provides a relative measure of muscle effort, while the GAIN-normalized data gives an estimate of the absolute muscle force. Therefore, the GAIN-normalized data tends to preserve the differences between subjects in the way they recruit their muscles to execute various tasks, while the MVC-normalized data will tend to suppress such differences. The appropriate choice of the EMG normalization method will depend on the specific question that an experimenter is attempting to answer.     

Passive and active mechanical properties of the superficial and deep digital flexor muscles in the forelimbs of anesthetized Thoroughbred horses

The superficial (SDF) and deep digital flexor (DDF) muscles are critical for equine forelimb locomotion. Knowledge of their mechanical properties will enhance our understanding of limb biomechanics. Muscle contractile properties derived from architectural-based algorithms may overestimate real forces and underestimate shortening capacity because of simplistic assumptions regarding muscle architecture. Therefore, passive and active (=total - passive) force-length properties of the SDF and DDF muscles were measured directly in vivo. Muscles from the right forelimbs of four Thoroughbred horses were evaluated during general anesthesia. Limbs were fixed to an external frame with the muscle attached to a linear actuator and load cell. Each muscle was stretched from an unloaded state to a range of prefixed lengths, then stimulated while held at that length. The total force did not exceed 4000 N, the limit for the clamping device. The SDF and DDF muscles produced 716+/-192 and 1577+/-203 N maximum active isometric force (F(max)), had ascending force-length ranges (R(asc)) of 5.1+/-0.2 and 9.1+/-0.4 cm, and had passive stiffnesses of 1186+/-104 and 1132+/-51 N/cm, respectively. The values measured for F(max) were much smaller than predicted based on conservative estimates of muscle specific tension and muscle physiological cross-sectional area. R(asc) were much larger than predicted based on muscle fiber length estimates. These data suggest that accurate prediction of the active mechanical behavior of architecturally complex muscles such as the equine DDF and SDF requires more sophisticated algorithms.     

Impedance, gas mixing, and bimodal ventilation in constricted lungs.

To evaluate the effect of increasing smooth muscle activation on the distribution of ventilation, lung impedance and expired gas concentrations were measured during a 16-breath He-washin maneuver in five nonasthmatic subjects at baseline and after each of three doses of aerosolized methacholine. Values of dynamic lung elastance (El,dyn), the curvature of washin plots, and the normalized slope of phase III (S(N)) were obtained. At the highest dose, El,dyn was 2.6 times the control value and S(N) for the 16th breath was 0.65 liter(-1). A previously described model of a constricted terminal airway was extended to include variable muscle activation, and the extended model was tested against these data. The model predicts that the constricted airway has two stable states. The impedances of the two stable states are independent of smooth muscle activation, but driving pressure and the number of airways in the high-resistance state increase with increasing muscle activation. Model predictions and experimental data agree well. We conclude that, as a result of the bistability of the terminal airways, the ventilation distribution in the constricted lung is bimodal.     

Pacific sleeper shark Somniosus pacificus trophic ecology in the eastern North Pacific Ocean inferred from nitrogen and carbon stable-isotope ratios and diet

Stable-isotope ratios of nitrogen (δ1?N) and lipid-normalized carbon (δ13C') were used to examine geographic and ontogenetic variability in the trophic ecology of a high latitude benthopelagic elasmobranch, the Pacific sleeper shark Somniosus pacificus. Mean muscle tissue δ13C' values of S. pacificus differed significantly among geographic regions of the eastern North Pacific Ocean. Linear models identified significant ontogenetic and geographic variability in muscle tissue δ1?N values of S. pacificus. The trophic position of S. pacificus in the eastern North Pacific Ocean estimated here from previously published stomach-content data (4·3) was within the range of S. pacificus trophic position predicted from a linear model of S. pacificus muscle tissue δ1?N (3·3-5·7) for fish of the same mean total length (L(T) ; 201·5 cm), but uncertainty in predicted trophic position was very high (95% prediction intervals ranged from 2·9 to 6·4). The relative trophic position of S. pacificus determined here from a literature review of δ1?N by taxa in the eastern North Pacific Ocean was also lower than would be expected based on stomach-content data alone when compared to fishes, squid and filter feeding whales. Stable-isotope analysis revealed wider variability in the feeding ecology of S. pacificus in the eastern North Pacific Ocean than shown by diet data alone, and expanded previous conclusions drawn from analyses of stomach-content data to regional and temporal scales meaningful for fisheries management.     

Tissue Turnover Rates and Isotopic Trophic Discrimination Factors in the Endothermic Teleost,Pacific Bluefin Tuna (Thunnus orientalis)

Stable isotope analysis (SIA) of highly migratory marine pelagic animals can improve understanding of their migratory patterns and trophic ecology. However, accurate interpretation of isotopic analyses relies on knowledge of isotope turnover rates and tissue-diet isotope discrimination factors. Laboratory-derived turnover rates and discrimination factors have been difficult to obtain due to the challenges of maintaining these species in captivity. We conducted a study to determine tissue- (white muscle and liver) and isotope- (nitrogen and carbon) specific turnover rates and trophic discrimination factors (TDFs) using archived tissues from captive Pacific bluefin tuna (PBFT), Thunnus orientalis, 1–2914 days after a diet shift in captivity. Half-life values for ¹⁵N turnover in white muscle and liver were 167 and 86 days, and for ¹³C were 255 and 162 days, respectively. TDFs for white muscle and liver were 1.9 and 1.1‰ for δ ¹⁵N and 1.8 and 1.2‰ for δ ¹³C, respectively. Our results demonstrate that turnover of ¹⁵N and ¹³C in bluefin tuna tissues is well described by a single compartment first-order kinetics model. We report variability in turnover rates between tissue types and their isotope dynamics, and hypothesize that metabolic processes play a large role in turnover of nitrogen and carbon in PBFT white muscle and liver tissues. ¹⁵N in white muscle tissue showed the most predictable change with diet over time, suggesting that white muscle δ ¹⁵N data may provide the most reliable inferences for diet and migration studies using stable isotopes in wild fish. These results allow more accurate interpretation of field data and dramatically improve our ability to use stable isotope data from wild tunas to better understand their migration patterns and trophic ecology.     

The titin N2B and N2A regions: biomechanical and metabolic signaling hubs in cross-striated muscles

Muscle specific signaling has been shown to originate from myofilaments and their associated cellular structures, including the sarcomeres, costameres or the cardiac intercalated disc. Two signaling hubs that play important biomechanical roles for cardiac and/or skeletal muscle physiology are the N2B and N2A regions in the giant protein titin. Prominent proteins associated with these regions in titin are chaperones Hsp90 and αB-crystallin, members of the four-and-a-half LIM (FHL) and muscle ankyrin repeat protein (Ankrd) families, as well as thin filament-associated proteins, such as myopalladin. This review highlights biological roles and properties of the titin N2B and N2A regions in health and disease. Special emphasis is placed on functions of Ankrd and FHL proteins as mechanosensors that modulate muscle-specific signaling and muscle growth. This region of the sarcomere also emerged as a hotspot for the modulation of passive muscle mechanics through altered titin phosphorylation and splicing, as well as tethering mechanisms that link titin to the thin filament system.     

The effects of N(omega)-propyl-L-arginine on reperfusion injury of skeletal muscle.

N(omega)-Propyl-L-arginine (NPA) is reported to be a highly selective inhibitor of neuronal nitric oxide synthase (nNOS). This in vivo study observed its role in ischemia/reperfusion (I/R) injury in rat skeletal muscle. Our results showed that NPA infusion significantly increased vessel diameters and blood flow in reperfused cremaster muscle, and slightly increased contractile function in reperfused extensor digitorum longus (EDL) muscle. In addition, NPA treatment slightly increased I/R-mediated downregulation of nNOS and eNOS mRNA and protein levels. Although NPA showed a beneficial role in I/R injury, our in vivo data do not support NPA as a selective nNOS inhibitor. Also, our data do not provide any insight into the mechanism of NPA. Thus, the in vivo mechanism of action of NPA needs to be further identified, and the role of nNOS in skeletal muscle I/R still remains to be determined.     

Which data should be tracked in forward-dynamic optimisation to best predict muscle forces in a pathological co-contraction case?

The choice of the cost-function for predicting muscle forces during a movement remains a challenge, especially in patients with neuromuscular disorders. Forward dynamics-based optimisations mainly track joint kinematics or torques, combined with a least-excitation criterion. Tracking marker trajectories and/or electromyography (EMG) has rarely been proposed. Our objective was to determine the best tracking objective-function to accurately predict the upper-limb muscle forces. A musculoskeletal model was created and EMG was simulated to obtain a reference movement – a shoulder abduction. A Gaussian noise (mean = 0; standard deviation = 15%) was added to the simulated EMG. Another noise – corresponding to the actual soft tissue artefacts (STA) of experimental shoulder abduction movements – was added to the trajectories of the markers placed on the model. Muscle forces were estimated from these noisy data, using forward dynamics assisted by six non-linear least-squared objective-functions. These functions involved the tracking of marker trajectories, joint angles or torques, with and without EMG-tracking. All six approaches used the same musculoskeletal model and were solved using a direct multiple shooting algorithm. Finally, the predicted joint angles, muscle forces and activations were compared to the reference values, using root-mean-square errors (RMSe) and biases. The force RMSe of the approach tracking both marker trajectories and EMG (18.45 ± 12.60 N) was almost five times lower than the one of the approach tracking only joint angles (82.37 ± 66.26 N) or torques (85.10 ± 116.40 N). Therefore, using EMG as a complementary tracking-data in forward dynamics seems to be promising for the estimation of muscle forces.     

Length-tension relation in Limulus striated muscle

Laser diffraction techniques coupled with simultaneous tension measurements were used to determine the length-tension relation in intact, small (0.5-mm thick, 10-mm wide, 20-25-mm long) bundles of a Limulus (horseshoe crab) striated muscle, the telson levator muscle. This muscle differs from the model vertebrate systems in that the thick filaments are not of a constant length, but shorten from 4.9 to approximately 2.0 micrometers as the sarcomeres shorten from 7 to 3 micrometers. In the Limulus muscle, the length-tension relation plateaued to an average maximum tension of 0.34 N/mm2 at a sarcomere length of 6.5 micrometers (Lo) to 8.0 micrometers. In the sarcomere length range from 3.8 to 12.5 micrometers, the muscle developed 50% or more of the maximum tension. When the sarcomere lengths are normalized (expressed as L/Lo) and the Limulus data are compared to those from frog muscle, it is apparent that Limulus muscle develops tension over a relatively greater range of sarcomere lengths.     

Effects of actin-myosin kinetics on the calcium sensitivity of regulated thin filaments

Activation of thin filaments in striated muscle occurs when tropomyosin exposes myosin binding sites on actin either through calcium-troponin (Ca-Tn) binding or by actin-myosin (A-M) strong binding. However, the extent to which these binding events contributes to thin filament activation remains unclear. Here we propose a simple analytical model in which strong A-M binding and Ca-Tn binding independently activates the rate of A-M weak-to-strong binding. The model predicts how the level of activation varies with pCa as well as A-M attachment, N·k(att), and detachment, k(det), kinetics. To test the model, we use an in vitro motility assay to measure the myosin-based sliding velocities of thin filaments at different pCa, N·k(att), and k(det) values. We observe that the combined effects of varying pCa, N·k(att), and k(det) are accurately fit by the analytical model. The model and supporting data imply that changes in attachment and detachment kinetics predictably affect the calcium sensitivity of striated muscle mechanics, providing a novel A-M kinetic-based interpretation for perturbations (e.g. disease-related mutations) that alter calcium sensitivity.     

Type I diabetes affects skeletal muscle glutamine uptake in a fiber-specific manner

Skeletal muscle serves as the body's major glutamine repository, and releases glutamine at enhanced rates during diabetes, but whether all muscles are equally affected is unknown. System N(m) activity mediates most trans-sarcolemmal glutamine movement, and although two System N (SN) isoforms have been identified (SN1/sodium-coupled neutral amino acid transporter or System N and A transporters [SNAT]-3; and SN2/SNAT5), their expression in skeletal muscle remains controversial. Here, the impact of Type I diabetes on glutamine uptake and System N transporter expression were examined in fast- and slow-twitch skeletal muscle from spontaneously diabetic (BB/Wor-DP) rats. Net glutamine uptake in fast-twitch fibers was decreased 75%-95%, but enhanced more than 2-fold in slow-twitch muscle from diabetic animals relative to nondiabetic controls. Both SNAT3 and SNAT5 mRNA were expressed in both muscle fiber types and their abundance was unaffected by diabetes. This represents the first report of differential fiber-specific effects of diabetes on skeletal muscle glutamine transport and the co-expression of distinct System N transporters in skeletal muscle.     

Metabolic activation of AMP kinase in vascular smooth muscle.

AMP-activated kinase (AMPK) is a highly conserved heterotrimeric kinase that functions as a metabolic master switch to coordinate cellular enzymes involved in carbohydrate and fat metabolism that regulate ATP conservation and synthesis. AMPK is activated by conditions that increase AMP-to-ATP ratio, such as exercise and metabolic stress. In the present study, we probed whether AMPK was expressed in vascular smooth muscle and would be activated by metabolic stress. Endothelium-denuded porcine carotid artery segments were metabolically challenged with 2-deoxyglucose (10 mM) plus N(2) (N(2)-2DG). These vessels exhibited a rapid increase in AMPK activity by 1 min that was near maximal by 20 min. AMPK inactivation on return to normal physiological saline was approximately 50% in 1 min and fully recovered by 5 min. Immunoprecipitation of the alpha(1)- and alpha(2)-catalytic subunit followed by immunoblot analysis for [P]Thr(172)-AMPK indicates that alpha(1)-AMPK accounts for all activity. Little if any alpha(2)-AMPK was detected in carotid smooth muscle. AMPK activity was not increased by contractile agonist (endothelin-1) or by the reported AMPK activators 5-aminoimidazole-4-carboxamide ribofuranoside (2 mM), metformin (2 mM), or phenformin (0.2 mM). AMPK activation by N(2)-2DG was associated with a rapid and pronounced reduction in endothelin-induced force and reduced phosphorylation of Akt and Erk 1/2. These data demonstrate that AMPK expression differs in vascular smooth muscle compared with striated muscles and that activation and inactivation after metabolic stress occur rapidly and are associated with signaling pathways that may regulate smooth-muscle contraction.     

Chemical modification of muscle protein in diabetes

Levels of glycation (fructose-lysine, FL) and advanced glycoxidation and lipoxidation end-products (AGE/ALEs) were measured in total skeletal (gastrocnemius) muscle and myofibril protein and compared to levels of the same compounds in insoluble skin collagen of control and diabetic rats. Levels of FL in total muscle and myofibril protein were 3-5% the level of FL in skin collagen. The AGE/ALEs, N(epsilon)-(carboxymethyl)lysine (CML) and N(epsilon)-(carboxyethyl)lysine, were also significantly lower in total muscle and myofibril protein, approximately 25% of levels in skin collagen. The newly described sulfhydryl AGE/ALE, S-(carboxymethyl)cysteine (CMC), was also measured in muscle; levels of CMC were comparable to those of CML and increased similarly in response to diabetes. Although FL and AGE/ALEs increased in muscle protein in diabetes, the relative increase was less than that seen in skin collagen. These data indicate that muscle protein is partially protected against the increase in both glycation and AGE/ALE formation in diabetes.     

Methods to collect, preserve, and prepare elasmobranch tissues for stable isotope analysis

Stable isotope analysis has the potential to expand our understanding of elasmobranch ecology. However, elasmobranchs share unique traits (i.e., retention of urea, lack of adipose tissue, cartilaginous skeletons) that require modified preparation techniques. Alternative tissue collection and preservation methods would allow sampling from ichthyology collections and at remote locations. We compared different collection, preservation, and preparation techniques to identify treatments that yielded robust isotopic data. Blood components collected in tubes coated with lithium heparin (an anti-coagulant) were not isotopically distinct from blood collected in no-additive tubes. Compared to frozen muscle, ethanol-treated muscle had altered ??¹³C values, but similar ??¹⁵N values. Finally, we removed lipids and urea with petroleum ether and deionized water, respectively. Although untreated and treated muscle had similar amino acid compositions, treated muscle preferentially lost ¹⁴N and had greater C:N ratios. These results indicate that urea affects isotope ratios and that water treatment removes urea without altering muscle protein composition. Although not exhaustive, our study begins to address the need for elasmobranch-specific methods.     

Lipid correction model of carbon stable isotopes for a cosmopolitan predator, spiny dogfish Squalus acanthias

Three lipid correction models were evaluated for liver and white dorsal muscle from Squalus acanthias. For muscle, all three models performed well, based on the Akaike Information Criterion value corrected for small sample sizes (AIC(c) ), and predicted similar lipid corrections to δ(13) C that were up to 2.8 ‰ higher than those predicted using previously published models based on multispecies data. For liver, which possessed higher bulk C:N values compared to that of white muscle, all three models performed poorly and lipid-corrected δ(13) C values were best approximated by simply adding 5.74 ‰ to bulk δ(13) C values.