Metabolic underpinnings of the paradoxical net phosphocreatine resynthesis in contracting rat gastrocnemius muscle

Net phosphocreatine (PCr) resynthesis during muscle contraction is a paradoxical phenomenon because it occurs under conditions of high energy demand. The metabolic underpinnings of this phenomenon were analyzed non-invasively using 31P-magnetic resonance spectroscopy in rat gastrocnemius muscle (n=11) electrically stimulated (7.6 Hz, 6 min duration) in situ under ischemic and normoxic conditions. During ischemic stimulation, [PCr] initially fell to a steady state (9+/-5% of resting concentration) which was maintained for the last 5 min of stimulation, whereas isometric force production decreased to a non-measurable level beyond 3 min. Throughout normoxic stimulation, [PCr] and force production declined to a steady state after respectively 1 min (5+/-3% of resting concentration) and 3.25 min (21+/-8% of initial value) of stimulation. Contrary to the observations under ischemia, a paradoxical net PCr resynthesis was recorded during the last 2 min of normoxic stimulation and was not accompanied by any improvement in force production. These results demonstrate that the paradoxical net PCr resynthesis recorded in contracting muscle relies exclusively on oxidative energy production and could occur in inactivated fibers, similarly to PCr resynthesis during post-exercise recovery.     

Metabolic underpinnings of the paradoxical net phosphocreatine resynthesis in contracting rat gastrocnemius muscle

Net phosphocreatine (PCr) resynthesis during muscle contraction is a paradoxical phenomenon because it occurs under conditions of high energy demand. The metabolic underpinnings of this phenomenon were analyzed non-invasively using ³¹P-magnetic resonance spectroscopy in rat gastrocnemius muscle (n=11) electrically stimulated (7.6 Hz, 6 min duration) in situ under ischemic and normoxic conditions. During ischemic stimulation, [PCr] initially fell to a steady state (9±5% of resting concentration) which was maintained for the last 5 min of stimulation, whereas isometric force production decreased to a non-measurable level beyond 3 min. Throughout normoxic stimulation, [PCr] and force production declined to a steady state after respectively 1 min (5±3% of resting concentration) and 3.25 min (21±8% of initial value) of stimulation. Contrary to the observations under ischemia, a paradoxical net PCr resynthesis was recorded during the last 2 min of normoxic stimulation and was not accompanied by any improvement in force production. These results demonstrate that the paradoxical net PCr resynthesis recorded in contracting muscle relies exclusively on oxidative energy production and could occur in inactivated fibers, similarly to PCr resynthesis during post-exercise recovery.     

Effects of hyperventilation on phosphocreatine kinetics and muscle deoxygenation during moderate-intensity plantar flexion exercise.

The effects of controlled voluntary hyperventilation (Hyp) on phosphocreatine (PCr) kinetics and muscle deoxygenation were examined during moderate-intensity plantar flexion exercise. Male subjects (n = 7) performed trials consisting of 20-min rest, 6-min exercise, and 10-min recovery in control [Con; end-tidal Pco(2) (Pet(CO(2))) approximately 33 mmHg] and Hyp (Pet(CO(2)) approximately 17 mmHg) conditions. Phosphorus-31 magnetic resonance and near-infrared spectroscopy were used simultaneously to monitor intramuscular acid-base status, high-energy phosphates, and muscle oxygenation. Resting intracellular hydrogen ion concentration ([H(+)](i)) was lower (P < 0.05) in Hyp [90 nM (SD 3)] than Con [96 nM (SD 4)]; however, at end exercise, [H(+)](i) was greater (P < 0.05) in Hyp [128 nM (SD 19)] than Con [120 nM (SD 17)]. At rest, [PCr] was not different between Con [36 mM (SD 2)] and Hyp [36 mM (SD 1)]. The time constant (tau) of PCr breakdown during transition from rest to exercise was greater (P < 0.05) in Hyp [39 s (SD 22)] than Con [32 s (SD 22)], and the PCr amplitude was greater (P < 0.05) in Hyp [26% (SD 4)] than Con [22% (SD 6)]. The deoxyhemoglobin and/or deoxymyoglobin (HHb) tau was similar between Hyp [13 s (SD 8)] and Con [10 s (SD 3)]; however, the amplitude was increased (P < 0.05) in Hyp [40 arbitrary units (au) (SD 23)] compared with Con [26 au (SD 17)]. In conclusion, our results indicate that Hyp-induced hypocapnia enhanced substrate-level phosphorylation during moderate-intensity exercise. In addition, the increased amplitude of the HHb response suggests a reduced local muscle perfusion in Hyp compared with Con.     

Asymmetric deformation of contracting human gastrocnemius muscle

Muscle fiber deformation is related to its cellular structure, as well as its architectural arrangement within the musculoskeletal system. While playing an important role in aponeurosis displacement, and efficiency of force transmission to the tendon, such deformation also provides important clues about the underlying mechanical structure of the muscle. We hypothesized that muscle fiber cross section would deform asymmetrically to satisfy the observed constant volume of muscle during a contraction. Velocity-encoded, phase-contrast, and morphological magnetic resonance imaging techniques were used to measure changes in fascicle length, pinnation angle, and aponeurosis separation of the human gastrocnemius muscle during passive and active eccentric ankle joint movements. These parameters were then used to subsequently calculate the in-plane muscle area subtended by the two aponeuroses and fascicles and to calculate the in-plane (dividing area by fascicle length), and through-plane (dividing muscle volume by area) thicknesses. Constant-volume considerations of the whole-muscle geometry require that, as fascicle length increases, the muscle fiber cross-sectional area must decrease in proportion to the length change. Our empirical findings confirm the definition of a constant-volume rule that dictates that changes in the dimension perpendicular to the plane, i.e., through-plane thickness, (-6.0% for passive, -3.3% for eccentric) equate to the reciprocal of the changes in area (6.8% for passive, 3.7% for eccentric) for both exercise paradigms. The asymmetry in fascicle cross-section deformation for both passive and active muscle fibers is established in this study with a ～22% in-plane and ～6% through-plane fascicle thickness change. These fiber deformations have functional relevance, not only because they affect the force production of the muscle itself, but also because they affect the characteristics of adjacent muscles by deflecting their line of pull.     

Influence of muscle shortening on the geometry of gastrocnemius medialis muscle of the rat

For static and dynamic conditions muscle geometry of the musculus gastrocnemius medialis of the rat was compared at different muscle lengths. The dynamic conditions differed with respect to isokinetic shortening velocity (25, 50 and 75 mm/s) of the muscle-tendon complex and in constancy of force (isotonic) and velocity (isokinetic) during shortening. Muscle geometry was characterized by fibre length and angle as well as aponeurosis length and angle. At high isokinetic shortening velocities (50 and 75 mm/s) small differences in geometry were found with respect to isometric conditions: aponeurosis lengths differed maximally by -2%, fibre length only showed a significant increase (+3.2%) at the highest shortening velocity. The isotonic condition only yielded significant differences of fibre angle (-4.5%) in comparison with isometric conditions. No significant differences of muscle geometry were found when comparing isotonic with isokinetic conditions of similar shortening velocity. The small differences of geometry between isometric and dynamic conditions are presumably due to the lower muscle force in the dynamic condition and the elastic behaviour of the aponeurosis. It is concluded that, unless very high velocities of shortening are used, the relationship between muscle geometry and muscle length in the isometric condition may be used to describe muscle geometry in the dynamic condition.     

Lactate dehydrogenase from gastrocnemius muscle of turtle

Five bands of lactate dehydrogenase (LDH) isoenzymes were seen by polyacrylamide gel electrophoresis in gastrocnemius muscle of the turtle (Kachuga smithi). The major band was of M2H2 type and was partially purified by gel filtration and affinity chromatography. The specific activity of the enzyme was 2.6 units/mg protein. The half-life of the enzyme at 4 degrees C, was about 7 days. The optimum temperature for enzyme activity was 30 degrees C and the enzyme was irreversibly inactivated at 40 degrees C. The optimum pH for the forward reaction (pyruvate to lactate) was 5.5, while for reverse reaction it was between 8.0 to 9.5. The apparent Km values for pyruvate, NADH, lactate and NAD+ were 0.20, 0.013, 25 and 0.333 mM, respectively. Oxalate was found to be the inhibitor of LDH with Ki of about 4.2 mM.     

High-energy phosphate transfer in human muscle: diffusion of phosphocreatine

The creatine kinase (CK) reaction is central to muscle energetics, buffering ATP levels during periods of intense activity via consumption of phosphocreatine (PCr). PCr is believed to serve as a spatial shuttle of high-energy phosphate between sites of energy production in the mitochondria and sites of energy utilization in the myofibrils via diffusion. Knowledge of the diffusion coefficient of PCr (D(PCr)) is thus critical for modeling and understanding energy transport in the myocyte, but D(PCr) has not been measured in humans. Using localized phosphorus magnetic resonance spectroscopy, we measured D(PCr) in the calf muscle of 11 adults as a function of direction and diffusion time. The results show that the diffusion of PCr is anisotropic, with significantly higher diffusion along the muscle fibers, and that the diffusion of PCr is restricted to a ～28-μm pathlength assuming a cylindrical model, with an unbounded diffusion coefficient of ～0.69 × 10(-3) mm(2)/s. This distance is comparable in size to the myofiber radius. On the basis of prior measures of CK reaction kinetics in human muscle, the expected diffusion distance of PCr during its half-life in the CK reaction is ～66 μm. This distance is much greater than the average distances between mitochondria and myofibrils. Thus these first measurements of PCr diffusion in human muscle in vivo support the view that PCr diffusion is not a factor limiting high-energy phosphate transport between the mitochondria and the myofibrils in healthy resting myocytes.     

Influence of gait velocity on gastrocnemius muscle fascicle behaviour during stair negotiation

The gastrocnemius medialis (GM) muscle plays an important role in stair negotiation. The aim of the study was to investigate the influence of cadence on GM muscle fascicle behaviour during stair ascent and descent. Ten male subjects (young adults) walked up and down a four-step staircase (with forceplates embedded in the steps) at three velocities (63, 88 and 116 steps/min). GM muscle fascicle length was measured using ultrasonography. In addition, kinematic and kinetic data of the lower legs, and GM electromyography (EMG) were measured. For both ascent and descent, the amount of fascicular shortening, shortening velocity, knee moment, ground reaction force and EMG activity increased monotonically with gait velocity. The ankle moment increased up to 88 steps/min where it reached a plateau. The lack of increase in ankle moment coinciding with further shortening of the fascicles can be explained by an increased shortening of the GM musculotendon complex (MTC), as calculated from the knee and ankle angle changes, between 88 and 116 steps/min only. For descent, the relative instant of maximum shortening, which occurred during touch down, was delayed at higher gait velocities, even to the extent that this event shifted from the double support to the single support phase.     

The kinetics of myofibrillar protein breakdown in perfused rat skeletal muscle

N^t-Methylhistidine, a non-reutilised amino acid present in some myofibrillar proteins, was radioactively labelled in vito with $\text{[}{}^{\mathrm{Me}}\text{-}{}^3\text{H}\text{]}\text{methionine}$. The specific radioactivities of protein-bound methylhistidine and free methylhistidine in perfusate after perfusion of rat hind limbs taken from prelabelled rats was determined. The decrease in urinary methylhistidine activity with time was determined for rats similarly labelled. Comparison of the specific activities of free and bound methylhistidine and the non-linear semilogarithmic plot of urinary methylhistidine activity suggest that the myofibrillar protein catabolism, as indicated by methylhistidine release, may not be a simple exponential process. The possibility of non-random decay is discussed and an alternative model proposed.     

Resonance in the human medial gastrocnemius muscle during cyclic ankle bending exercise.

We investigated the behavior of the muscle tendon unit (MTU) of the medial gastrocnemius muscle during cyclic ankle bending exercise at eight different frequencies (ranging from 1.33 to 3.67 Hz). The changes in the length of fascicle in the muscle during the exercises were determined by real-time ultrasound imaging. The coordinates of anatomical references and the ground reaction force were determined from video recording and a force plate, respectively. The length change of the MTU (the distance from the origin to insertion of the muscle) was calculated from changes in the knee and ankle joint angles. It was found that the amplitude ratio and phase difference between the fascicle and MTU lengths were both dependent on the movement frequency. At lower frequencies, the fascicle lengths varied almost in phase with the MTU length, whereas they varied out of phase at the higher frequencies. At intermediate frequency, the amplitude of the fascicle became very small compared with that of the MTU, which is considered resonance. We constructed a mechanical model of the MTU based on a notion of forced oscillation in a mass-spring system. The obtained data were well explained by the model. It was concluded that the behavior of the MTU highly depends on the movement frequency due to the viscoelasticity of the MTU.     

Influence of tendon slack on electromechanical delay in the human medial gastrocnemius in vivo.

The purpose of this study was to clarify the influence of muscle-tendon complex stretch on electromechanical delay (EMD) in terms of the extent of tendon slack in the human medial gastrocnemius (MG). EMD and MG tendon length were measured at each of five ankle joint angles (-30, -20, -10, 0, and 5 degrees : positive values for dorsiflexion) using percutaneous electrical stimulation and ultrasonography, respectively. The extent of MG tendon slack was calculated as MG tendon length shortening, standardized with MG tendon slack length obtained at the joint angle (-16 degrees +/- 5 degrees ) where the passive ankle joint torque was zero. EMD at -30 degrees (19.2 +/-2.2 ms) and -20 degrees (17.2 +/- 1.3 ms) was significantly greater than that at -10 degrees (16.0 +/-2.3 ms), 0 degrees (15.0 +/-1.4 ms), and 5 degrees (14.8 +/-1.4 ms), and at 0 and 5 degrees, respectively. The relative EMD, normalized with the maximal EMD for each subject, decreased dependent on the extent of decrease in MG tendon slack. There were no significant differences in EMD among the joint angles (-10, 0, and 5 degrees ) where MG tendon slack was taken up. These results suggest that the extent of tendon slack is an important factor for determining EMD.     

Characterization of cationic liposomes. Influence of the bilayer composition on the kinetics of the liposome breakdown

The cationic large unilamellar mixed liposomes from 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and didodecyldimethylammonium bromide (DDAB) or dioctadecyldimethylammonium bromide (DODAB) were prepared. The influence of the addition of Triton X-100 (TX-100) or octaethylene glycol mono-n-dodecylether (C₁₂E₈) on the membrane integrity was investigated turbidimetrically. The stability of the liposomal systems was estimated by monitoring fluorimetrically at 25 °C the rate of spontaneous and surfactant-induced release of entrapped 5(6)-carboxyfluorescein (CF). In order to evaluate the interaction of the cationic DODAB guest with the host POPC membrane, the main phase transition temperatures (T_m) were determined by electron paramagnetic resonance spectroscopy (EPR). All the results obtained show that the presence of DODAB and DDAB stabilizes the POPC liposomes. The extent of stabilization depends on the concentration and nature of the cationic guest.     

In vivo passive mechanical properties of the human gastrocnemius muscle belly

The purpose of the present study was to determine the in vivo passive mechanical properties, including the length below the slack length, of the gastrocnemius muscle (GAS) belly in humans. Transverse ultrasound images of the medial head of the GAS were taken in 11 subjects during passive knee extension from 80 degrees to 5 degrees with a constant ankle joint angle of 10 degrees (0 degrees is the neutral ankle position: positive values for dorsiflexion). The change in passive ankle joint moment (Mp), which is produced only by the GAS length change, was also measured during passive knee extension. The onset of Mp during passive knee extension was found to be 43+/-8 degrees (mean+/-SD) when the baseline of the Mp was set at the average Mp in the range of 55-60 degrees where the Mp was almost constant (SD<0.03 Nm). At this onset, the muscle fascicle length of the GAS (Lf) was 46+/-7 mm (slack length; Lfs). Lf at 80 degrees was 6+/-4 mm (13+/-6%) less than the Lfs, and Lf at 5 degrees was 12+/-5 mm (27+/-11%) greater than the Lfs. The passive force-resisting compression of the GAS did not produce a dorsiflexion moment in the joint angle range adopted. The passive ankle joint moment increased linearly with Lf (coefficient of determination (R2)=0.85-0.96), and the slopes of the relationships between Lf and Mp, and between the relative Lf to Lfs and Mp were 0.093+/-0.038 Nm/mm and 0.043+/-0.021 Nm/%Lfs. The findings of the present study can be implemented in musculoskeletal modeling, which would provide a more accurate evaluation of the passive mechanical properties of muscle during movement.     

Influence of muscle dimensions on economy of isometric exercise in rat medial gastrocnemius muscles in situ

The effect of muscle dimensions on economy (force-time integral divided by the amount of energy utilized) was investigated in male rats (body mass range 95-490 g), anaesthetized with pentobarbital. The medial gastrocnemius muscle in situ performed 6 maximal isometric contractions of 350 ms duration (1.s-1) at twitch optimum length at 35 degrees C. The areas under the 6 time-force curves were added to obtain force-time integral of the experiment. Differences of concentrations of ATP, phosphocreatine and lactate between experimental and contralateral (resting) muscles were used to calculate high-energy phosphate consumption due to stimulation. Muscle mass and cross-sectional area increased (approximately +400% and +300%, respectively) over the rat body mass range studied. Muscle length and length of the most distal fibre bundle increased by approximately 17 mm and 4 mm, respectively. Force-time integral (N.s) increased proportional to cross-sectional area whereas high-energy phosphate consumption (mumoles) increased proportional to muscle mass. The relative fraction of the total energy consumption utilized for force-independent processes was independent of rat body mass. The economy of the actomyosin system was unaffected during growth, whereas economy of the whole muscle decreased during growth by approximately 30% (p less than 0.001). The effect of muscle dimensions on economy is discussed with respect to human endurance capacity measured by voluntary isometric contractions.