A 31P-n.m.r. study of the acute effects of beta-blockade on the bioenergetics of skeletal muscle during contraction.

1. The effects of beta-adrenoceptor antagonist administration on skeletal muscle contractile performance and bioenergetics in vivo have been investigated during unilateral sciatic nerve stimulation in the rat. 2. Two muscle stimulation protocols have been used: supramaximal stimulation at 4 Hz, or incremental supramaximal stimulation at 1, 2 and 4 Hz. Changes in high-energy phosphate concentrations were followed using 31P-n.m.r., and gastrocnemius muscle twitch characteristics were monitored continuously. 3. Under all conditions investigated, DL-propranolol administration (2.5 mg/kg body wt.) caused a significant decrease in cyclic AMP concentrations in resting and stimulated gastrocnemius muscle, prevented an increase in heart rate upon muscle stimulation, but did not affect plasma glucose, fatty acid or lactate concentrations in comparison with values obtained in control experiments. 4. Administration of DL-propranolol 5 min or 35 min before unilateral stimulation of 4 Hz had no effect on changes in muscle phosphocreatine, ATP or Pi concentrations, intracellular pH or contractile performance. 5. In contrast, animals receiving DL-propranolol 5 min before unilateral stimulation of 1, 2 and 4 Hz showed a significant deterioration in gastrocnemius muscle tension development during 2 and 4 Hz stimulation compared with control animals. Concurrent with this change in contractile performance was a higher muscle concentration of phosphocreatine, a lower concentration of Pi and no significant change in intramuscular pH compared with control experiments. 6. The changes in muscle performance and bioenergetics observed during the incremental stimulation protocol were not observed when D-propranolol was administered and could be completely circumvented by a short period of muscle stimulation of 4 Hz prior to initiation of the incremental stimulation protocol. 7. Mechanisms are discussed which may account for the failure of gastrocnemius muscle to generate the expected force during the incremental stimulation protocol in the presence of beta-blockade.     

31P-NMR studies of phosphate metabolites in intact red and white swimming muscles of cod (Gadus morhua L.)

31P-NMR was used to characterise intracellular phosphate pools and their post mortem changes at 7 degrees C in intact red and white cod muscles under anaerobic conditions. A total phosphate content of 55 and 60 mM was observed in red and white muscle, respectively. The concentration of P-creatine was 14 mM in the white and 9 mM in the red muscle, while that of inorganic phosphate, Pi (30 mM), ATP (9 mM), and sugar phosphate (5 mM) were similar in both muscles. During the first 90 min after death, the decrease in P-creatine showed a first order breakdown with a concomitant stoichiometric increase in Pi content, whereas the ATP and sugar phosphate remained the same. The intracellular pH decreased from 7.4 to 7.3 in this period. The steady-state rate constant of myosin ATPase was 0.0054 and 0.0022/min for red and white muscles, respectively. Individuals kept under diminished oxygen tension prior to being killed, showed a reduced P-creatine level in both muscles.     

Molecular transformations in sarcoplasmic reticulum of fast-twitch muscle by electro-stimulation.

Chronic electro-stimulation of fast-twitch rabbit muscle with the frequency pattern received by a slow-twitch muscle induces a progressive transformation of the sarcoplasmic reticulum. After 2 days stimulation activities of Ca2+-dependent ATPase and of Ca2+ transport begin to decrease, and are paralleled by a progressive decrease in Ca2+-dependent and Ca2+, Mg2+-dependent phosphoprotein formation, reduced rate of dephosphorylation and a rearrangement of the electrophoretic polypeptide and phosphoprotein patterns. These findings suggest a transformation of the sarcoplasmic reticulum to resemble that of a slow-twitch muscle. This transformation is paralleled by increase in time-to-peak of twitch contraction and half relaxation time and occurs before conversion of the myosin light chain pattern is observed. The parallel time course of changes in contractile properties of stimulated muscle and the molecular and functional properties of the sarcoplasmic reticulum emphasizes the definitive role of the latter in determining the twitch characteristics of fast and slow twitch muscles.     

Evaluation of exercise muscle energetics by NMR

Magnetic resonance imaging (MRI) is superior to ultrasonography and X-CT especially in density resolution in soft tissue. 31P NMR provides information on metabolism, which has not been obtained in vivo by conventional methods, such as phosphocreatine (PCr), inorganic phosphate (Pi), ATP, and intracellular pH. We used MRI and 31P NMR spectroscopy to study skeletal muscle metabolism of human and rat. These NMR results suggested that 1) estimation of muscle fiber composition, 2) evaluation of muscle ATP turnover and 3) imaging of local muscle fatigue are possible.     

Altered Energetics of Exercise Explain Risk of Rhabdomyolysis in Very Long-Chain Acyl-CoA Dehydrogenase Deficiency

Rhabdomyolysis is common in very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) and other metabolic myopathies, but its pathogenic basis is poorly understood. Here, we show that prolonged bicycling exercise against a standardized moderate workload in VLCADD patients is associated with threefold bigger changes in phosphocreatine (PCr) and inorganic phosphate (Pi) concentrations in quadriceps muscle and twofold lower changes in plasma acetyl-carnitine levels than in healthy subjects. This result is consistent with the hypothesis that muscle ATP homeostasis during exercise is compromised in VLCADD. However, the measured rates of PCr and Pi recovery post-exercise showed that the mitochondrial capacity for ATP synthesis in VLCADD muscle was normal. Mathematical modeling of oxidative ATP metabolism in muscle composed of three different fiber types indicated that the observed altered energy balance during submaximal exercise in VLCADD patients may be explained by a slow-to-fast shift in quadriceps fiber-type composition corresponding to 30% of the slow-twitch fiber-type pool in healthy quadriceps muscle. This study demonstrates for the first time that quadriceps energy balance during exercise in VLCADD patients is altered but not because of failing mitochondrial function. Our findings provide new clues to understanding the risk of rhabdomyolysis following exercise in human VLCADD.     

THE SARCOPLASMIC RETICULUM, THE T SYSTEM, AND THE MOTOR TERMINALS OF SLOW AND TWITCH MUSCLE FIBERS IN THE GARTER SNAKE

Twitch and slow muscle fibers, identified morphologically in the garter snake, have been examined in the electron microscope. The transverse tubular system and the sarcoplasmic reticulum are separate entities distinct from each other. In twitch fibers, the tubular system and the dilated sacs of the sarcoplasmic reticulum form triads at the level of junction of A and I bands. In the slow fibers, the sarcoplasmic reticulum is severely depleted in amount and the transverse tubular system is completely absent. The junctional folds of the postsynaptic membrane of the muscle fiber under an "en grappe" ending of a slow fiber are not so frequent or regular in occurrence or so wide or so long as under the "en plaque" ending of a twitch fiber. Some physiological implications of these differences in fine structure of twitch and slow fibers are discussed. The absence of the transverse tubular system and reduction in amount of sarcoplasmic reticulum, along with the consequent disposition of the fibrils, the occurrence of multiple nerve terminals, and the degree of complexity of the post junctional folds of the sarcolemma appear to be the morphological basis for the physiological reaction of slow muscle fibers.     

31P NMR study of phosphorus metabolites in fast and slow muscles

1. 31P NMR was used to characterize phosphate pools in perchloric acid extracts of muscles with various composition of muscle fibre types. 2. The white m. pectoralis major (MPM) of chickens 15 min post mortem is characterized by 1.6-times higher relative content of phosphocreatine (PCr) in comparison with mixed leg muscle (LM) of this species. The glycerophosphorylcholine (GPC) does not occur in MPM at NMR detectable level in contrast to the leg muscles. Relative amounts of other phosphates are similar in both muscles. 3. The intermediate MPM of pigeons as well as mixed LM of this species contain 15 min post mortem a very small amount of PCr and ATP but a large amount of inorganic phosphate. Relative content of GPC is higher in leg muscles than in intermediate MPM. 4. Muscles with higher occurrence of white fibres contain relatively more PCr than muscles with lower occurrence of white fibres. 5. The occurrence of GPC seems to be connected with metabolism of red muscle fibres.     

31P-NMR spectroscopy and the metabolic properties of different muscle fibers

To study the in vivo recruitment of different fiber types and their metabolic properties, 31P-nuclear magnetic resonance spectroscopy (31P-NMRS) of the human calf muscle was performed in seven normal sedentary subjects. In the exhaustive exercise protocol used, the work load was increased every minute during 5 min. This resulted in a prominent split of the Pi resonance in all subjects, indicating pH compartmentation in the muscles studied. From the chemical shift of the Pi peaks relative to phosphocreatine (PCr) at the end of the exercise, intracellular pH (pHi) averaged 6.92 +/- 0.05 (SD) in compartment 1 and 6.23 +/- 0.15 in compartment 2. The recovery of both Pi resonances after exercise could be followed easily in five of these subjects. The recovery rate of the Pi peak is a good estimate of the oxidative metabolism at the end of the exercise. A monoexponential regression analysis showed that the mean initial recovery rate S0 was 2.49 +/- 0.17%/s in compartment 1 and only 0.87 +/- 0.12%/s in compartment 2, indicating aerobic function three times higher in compartment 1 at the end of exercise. The mean relative ATP fraction dropped significantly (P less than 0.001), from 20.0 +/- 1.0% of the total 31P signal integral before exercise to 14.0 +/- 1.6% at the end of exercise. The simultaneous visualization of two compartments, in good order, one with high pHi and fast recovery and another with low pHi and slow recovery, is rationalized by the different metabolic behavior of type I and II fibers in human calf muscle in response to exhaustive exercise. This study demonstrates that 31P-NMRS is an excellent noninvasive procedure to quantify aerobic metabolism in both fiber types simultaneously.     

Sexually differentiated,androgen-regulated,larynx-specific myosin heavy-chain isoforms in <Emphasis Type="Italic">Xenopus tropicalis</Emphasis>; comparison to <Emphasis Type="Italic">Xenopus laevis</Emphasis>

We have shown that the sarcoplasmic myosin heavy-chain (MyHC) isoform xtMyHC-101d is highly and specifically expressed in the larynx of the aquatic anuran, Xenopus tropicalis. In male larynges, the predominant MyHC isoform is xtMyHC-101d, while in females, another isoform, xtMyHC-270c, predominates. The X. tropicalis genome has been sequenced in its entirety, and xtMyHC-101d is part of a specific array of xtMyHC genes expressed otherwise in embryonic muscles (Nasipak and Kelley, Dev Genes Evol, in press, 2008). The administration of the androgen dihydrotestosterone increases the expression of xtMyHC-101d in juvenile larynges of both sexes. Using ATPase histochemistry, we found that in adults, X. tropicalis male laryngeal muscle contains only fast-twitch fibers, while the female laryngeal muscle contains a mix of fast- and slow-twitch fibers. Juvenile larynges are female-like in fiber type composition (44% slow twitch, 56% fast twitch); androgen treatment increases the percentage of fast-twitch fibers to 86%. xtMyHC-101d predominates in larynges of dihydrotestosterone-treated juveniles but not in larynges of untreated juveniles. We compared the larynx-specific expression of xtMyHC genes in X. tropicalis to the MyHC gene expressed in X. laevis larynx (xlMyHC-LM) by sequencing the entire xlMyHC-LM gene. The androgen-regulated xtMyHC that predominates in the male larynx of X. tropicalis is not the gene phylogenetically most similar to xlMyHC-LM at the nucleotide level but is instead a similar isoform found in the same MyHC array and expressed in the embryonic muscle.     

Platelet-derived-growth-factor-stimulated heterogeneous polyphosphoinositide metabolism and phosphate uptake in C3H fibroblasts.

1. Gated 31P-n.m.r. spectra were obtained from the ankle flexor muscles of the rat at various times after 3 s isometric tetanic contraction. This allowed the time course of changes in phosphocreatine (PCr), Pi and free ADP concentrations and intracellular pH to be monitored in skeletal muscle in vivo with 1 s time resolution. 2. ATP concentration did not change significantly, either during the recovery from a 3 s tetanus or during the overall protocol. 3. The calculated rate of recovery of ADP towards pre-stimulation levels was very rapid (t1/2 less than 5 s). The rate of Pi disappearance (t1/2 = 14 s) was more rapid than the rate of PCr synthesis (t1/2 = 24 s), resulting in a significant transient decrease in n.m.r.-visible PCr + Pi between 25 and 45 s after tetanic contraction. 4. The rates of PCr, Pi and ADP recovery are higher than those previously reported for recovery from steady-state exercise in humans or twitch isometric contraction in animals.     

Analysis of fiber types in the pigeon's metapatagialis muscle II. Effects of denervation

The pigeon's metapatagialis muscle consists of three slips, two twitch and one tonic, and these slips are distinguishable at the gross anatomical level. Comparative studies of denervation are facilitated because the two fiber types are under the same mechanical forces, can be denervated as one muscle, and can be distinguished after denervation. Both fiber types atrophied after denervation, with the twitch fibers having a more variable response. Pathological alterations observed by light microscopy suggested that the twitch fibers were more affected by denervation than the tonus fibers. Ultrastructurally, both fiber types showed the same changes, with the twitch fibers again being more consistently altered. Proliferation of the transverse tubular system and sarcoplasmic reticulum were more marked in the tonus than twitch fibers, and the sarcoplasmic reticulum proliferated prior to the transverse tubules. Filament and fibril degeneration, peripheral and central degeneration, lysosomes and their derivatives, and satellite cell proliferation were common to both fiber types. Contracture knots were common to the denervated fibers, and were suggested to be characteristic of degenerating fibers. Degenerating motor end plates were observed, and most neurons in the fibers were naked, lacking myelin sheaths. The results are discussed in relation to the function of the neuron in maintaining the muscle, and the possibility of denervation inducing a transformation of tonic to twitch fibers.     

Training-induced skeletal muscle adaptations are independent of systemic adaptations

To isolate the peripheral adaptations to training, five normal subjects exercised the nondominant (ND) wrist flexors for 41 +/- 11 days, maintaining an exercise intensity below the threshold required for cardiovascular adaptations. Before and after training, intracellular pH and the ratio of inorganic phosphate to phosphocreatine (Pi/PCr) were measured by 31P magnetic resonance spectroscopy. Also maximal O2 consumption (VO2 max), muscle mass, and forearm blood flow were determined by graded systemic exercise, magnetic resonance imaging, and venous occlusion plethysmography, respectively. Blood flow, Pi/PCr, and pH were measured in both forearms at rest and during submaximal wrist flexion at 5, 23, and 46 J/min. Training did not affect VO2 max, exercise blood flow, or muscle mass. Resting pH, Pi/PCr, and blood flow were also unchanged. After training, the ND forearm demonstrated significantly lower Pi/PCr at 23 and 46 J/min. Endurance, measured as the number of contractions to exhaustion, also was increased significantly (63%) after training in the ND forearm. We conclude that 1) forearm training results in a lower Pi/PCr at identical submaximal work loads; 2) this improvement is independent of changes in VO2 max, muscle mass, or limb blood flow; and 3) these differences are associated with improved endurance and may reflect improved oxidative capacity of skeletal muscle.     

Diazepam reveals different rate-limiting processes in rat skeletal muscle contraction

The action of the tranquilizer diazepam on rat skeletal muscle showed that relaxation of isometric twitches is controlled by different processes in extensor digitorum longus (fast-twitch) and soleus (slow-twitch) muscles. Diazepam caused an increase in the amplitude of twitches in fibres from both muscles but increased the twitch duration only in soleus. The amplitude of fused tetani were reduced in both muscles and the rate of relaxation after the tetanus slowed by as much as 34% when the amplitude of the tetanus was reduced by only 11%. The slower tetanic relaxation indicated that calcium uptake by the sarcoplasmic reticulum was slower than normal in slow- and fast-twitch fibres. We conclude therefore that calcium uptake by the sarcoplasmic reticulum is rate limiting for twitch relaxation in slow-twitch but not fast-twitch fibres and suggest that calcium binding to parvalbumin controls relaxation in the fast fibres.     

Forearm metabolic asymmetry detected by 31P-NMR during submaximal exercise

This study evaluated the relationship of skeletal muscle energy metabolism to forearm blood flow and muscle mass in the dominant (D) and nondominant (ND) forearms of normal subjects. 31P-Magnetic resonance spectroscopy was used to determine intracellular pH and the ratio of inorganic phosphate to phosphocreatine (Pi/PCr), an index of energy metabolism. Forearm blood flow and muscle mass were measured by venous occlusion plethysmography and magnetic resonance imaging, respectively. Metabolic measurements and flow were determined at rest and during submaximal exercise in both forearms. After a warm-up period, six normal right-handed male subjects performed 7.5 min of wrist flexion exercise in the magnet (1 contraction every 5 s), first with the ND forearm and then with the D forearm, at 23, 46, and 69 J/min. At rest, there were no differences between forearms in Pi/PCr or pH. However, at each work load the D forearm demonstrated significantly lower Pi/PCr and higher pH than the ND forearm. Blood flow was not significantly different between the forearms at rest or during exercise. Because these subjects were not engaged in unilateral arm training, we conclude that 1) Pi/PCr is lower and pH is higher in the D compared with the ND forearm in normal subjects during submaximal exercise, 2) these differences are independent of muscle mass and blood flow, and 3) the cumulative effect of long-term, low-level daily activity provides an adequate training stimulus for muscular metabolic adaptations.     

Application of high-field 1H-NMR spectroscopy for the study of perifused amphibian and excised mammalian muscles

Frog sartorius and gastrocnemius muscles were perifused at 20 degrees C, the intracellular pH (pHi) and the concentration of phosphocreatine were determined in the resting muscle by 1H-NMR spectroscopy at 470 MHz; values of pHi = 7.31 +/- 0.05 (n = 7) and concentration of phosphocreatine = 20.4 +/- 1.1 mumol/g wet wt. (n = 6) were found. The hydrolysis of phosphocreatine and the simultaneous increase in lactate upon perifusion with 10 mM caffeine (in Ringer's solution) was followed with a time resolution of 1 min. Lactate increased at a rate of 1.0 mumol/g per min, but no pHi change was recorded during the time monitored. The lower limit for the buffering capacity of the muscle cytosol was estimated to be 16.7 mumol/g muscle per pH unit from the uncertainty in pHi determination (+/- 0.03 pH units) and from the amount of lactate produced and phosphocreatine hydrolyzed. Changes in pHi, lactate concentration and fatty acyl chain intensity were monitored by 1H-NMR spectroscopy at 361 MHz in ischemic rat skeletal muscle, excised and stored at 20 degrees C. The resonances in the 1H-NMR spectrum of a human skeletal muscle perchloric acid extract are reported and tentatively assigned.     

Effect of Dietary Magnesium on Post Mortem Phosphocreatine Utilization in Skeletal Muscle of Swine: A Non-Invasive Study Using 31P-NMR Spectroscopy

The effect of dietary magnesium on the post mortem PCr (phosphocreatine) decay in muscle of heterozygote malignant hyperthermia pigs was studied after in vivo exposure to a combination of halothane and succinylcholine. The pigs were anaesthetized with halothane and succinylcholine was injected in the ear vein. Immediately after initiation of the depolarizing neuromuscular blocking effect of succinylcholine the animals were captive-bolt stunned. The PCr decay, reflecting ATP turnover, was followed in situ by 31P-NMR spectroscopy in the biceps femoris muscle for the subsequent 40-70 min post mortem. In 3 of the 4 experiments, the Mg-fed pig had a significantly reduced rate of PCr hydrolysis compared to the control animal. The mechanism of this magnesium effect is unknown.     

Morphological and biochemical correlates of skeletal muscle contractility in the cat. II. Physiological and biochemical studies.

Isometric twitch characteristics and biochemical parameters of isolated myosin and sarcoplasmic reticulum have been compared in three cat hind limb muscles. The fast twitch caudofemoralis and the slow twitch soleus are almost pure muscles as judged from histochemical studies. Isolated myosin from the caudofemoralis is not only 2- to 3-fold higher in its ATPase activities than that of the soleus, but also in non-dissociated forms has greater electrophoretic mobility than the soleus myosin. Purified myosins from fast muscles as well as soleus exhibited three light chains upon electrophoresis. However, the intact non-solubilized myosins differed in electrophoretic mobilities. The sarcoplasmic reticulum fraction isolated from caudfemoralis exhibits faster rates of Ca++ binding and uptake than soleus, and when fit to a two component model, the caudofemoralis SR exhibits a higher amount of a fast binding site than does soleus SR, features reflected in differences in the relaxation time of the two muscles. In contrast, the fast twitch tibialis anterior has been shown to be a gradient of fiber types and its isometric twitch may be separated by selective nerve stimulation, into a fast and a slow twitch component. Our findings that myosin fractions, as well as sarcoplasmic reticulum fractions isolated from these two components differ with respect to their biochemical characteristics add support to the possibility of a dual function in this muscle.     

Ultrastructure of muscle fibers of an extraocular muscle of the pigeon

An electron microscopic analysis of the internal rectus muscle of the eye of the pigeon permitted identification of three types of muscle fibers: the first type shows the features previously described in vertebrate twitch fibers. The second type has very scarce sarcoplasmic reticulum at the A-band, their myofibrils fuse together at this level; the Z-line is large and the M-line is not present; the thick filaments are more abundant per unit area than in the first type of fibers, their hexagonal array is slightly disrupted and the fibers appear more opaque than the other two fiber types. The third type of fibers has bundles of myofibrils incompletely surrounded by sarcoplasmic reticulum at the A-band; the Z-line is large; the M-line is present and the hexagonal array of the thick filaments is maintained.     

Effects of fatigue on sarcoplasmic reticulum and myofibrillar properties of rat single muscle fibers.

Force decline during fatigue in skeletal muscle is attributed mainly to progressive alterations of the intracellular milieu. Metabolite changes and the decline in free myoplasmic calcium influence the activation and contractile processes. This study was aimed at evaluating whether fatigue also causes persistent modifications of key myofibrillar and sarcoplasmic reticulum (SR) proteins that contribute to tension reduction. The presence of such modifications was investigated in chemically skinned fibers, a procedure that replaces the fatigued cytoplasm from the muscle fiber with a normal medium. Myofibrillar Ca(2+) sensitivity was reduced in slow-twitch muscle (for example, the pCa value corresponding to 50% of maximum tension was 6.23 +/- 0.03 vs. 5.99 + 0.05, P < 0.01, in rested and fatigued fibers) and not modified in fast-twitch muscle. Phosphorylation of the regulatory myosin light chain isoform increased in fast-twitch muscle. The rate of SR Ca(2+) uptake was increased in slow-twitch muscle fibers (14.2 +/- 1.0 vs. 19.6 +/- 2. 5 nmol. min(-1). mg fiber protein(-1), P < 0.05) and not altered in fast-twitch fibers. No persistent modifications of SR Ca(2+) release properties were found. These results indicate that persistent modifications of myofibrillar and SR properties contribute to fatigue-induced muscle force decline only in slow fibers. These alterations may be either enhanced or counteracted, in vivo, by the metabolic changes that normally occur during fatigue development.     

Phosphorylation in vivo of the P light chain of myosin in rabbit fast and slow skeletal muscles.

The P light chain of myosin is partially phosphorylated in resting slow and fast twitch skeletal muscles of the rabbit in vivo. The extent of P light-chain phosphorylation increases in both muscles on stimulation. Rabbit slow-twitch muscles contain two forms of the P light chain that migrate with the same electrophoretic mobilities as the two forms of P light chain in rabbit ventricular muscle. The rate of phosphorylation of the P light chain in slow-twitch muscle is slower than its rate of phosphorylation in fast-twitch muscles during tetanus. The rate of P light-chain dephosphorylation is slow after tetanic contraction of fast-twitch muscles in vivo. The time course of dephosphorylation does not correlate with the decline of post-tetanic potentiation of peak twitch tension in rabbit fast-twitch muscles. The frequency of stimulation is an important factor in determining the extent of P light-chain phosphorylation in fast- and slow-twitch muscles.