E-C coupling failure in mouse EDL muscle after in vivo eccentric contractions

The objectives of this research were to determine thecontribution of excitation-contraction (E-C) coupling failure to the decrement in maximal isometric tetanic force(P_o) in mouse extensor digitorumlongus (EDL) muscles after eccentric contractions and to elucidatepossible mechanisms. The left anterior crural muscles of femaleICR mice (n = 164) wereinjured in vivo with 150 eccentric contractions.P_o, caffeine-,4-chloro-m-cresol-, andK⁺-induced contracture forces,sarcoplasmic reticulum (SR) Ca²⁺release and uptake rates, and intracellularCa²⁺ concentration([Ca²⁺]_i)were then measured in vitro in injured and contralateral control EDLmuscles at various times after injury up to 14 days. On the basis ofthe disproportional reduction inP_o (~51%) compared with caffeine-induced force (~11-21%), we estimate that E-C coupling failure can explain 57-75% of theP_o decrement from 0 to 5 days postinjury. Comparable reductions inP_o andK⁺-induced force (51%), and minorreductions (0-6%) in the maximal SRCa²⁺ release rate, suggest thatthe E-C coupling defect site is located at the t tubule-SR interfaceimmediately after injury. Confocal laser scanning microscopy indicatedthat resting[Ca²⁺]_iwas elevated and peak tetanic[Ca²⁺]_iwas reduced, whereas peak4-chloro-m-cresol-induced[Ca²⁺]_iwas unchanged immediately after injury. By 3 days postinjury, 4-chloro-m-cresol-induced[Ca²⁺]_ibecame depressed, probably because of decreased SRCa²⁺ release and uptake rates(17-31%). These data indicate that the decrease inP_o during the first several daysafter injury primarily stems from a failure in the E-C couplingprocess.

     

Rectification of skeletal muscle ryanodine receptor mediated by FK506 binding protein.

The cytosolic receptor for immunosuppressant drugs, FK506 binding protein (FKBP12), maintains a tight association with ryanodine receptors of sarcoplasmic reticulum (SR) membrane in skeletal muscle. The interaction between FKBP12 and ryanodine receptors resulted in distinct rectification of the Ca release channel. The endogenous FKBP-bound Ca release channel conducted current unidirectionally from SR lumen to myoplasm; in the opposite direction, the channel deactivated with fast kinetics. The binding of FKBP12 is likely to alter subunit interactions within the ryanodine receptor complex, as revealed by changes in conductance states of the channel. Both on- and off-rates of FKBP12 binding to the ryanodine receptor showed clear dependence on the membrane potential, suggesting that the binding sites of FKBP12 reside in or near the conduction pore of the Ca release channel. Rectification of the Ca release channel would prevent counter-current flow during the rapid release of Ca from SR membrane, and thus may serve as a negative feedback mechanism that participates in the process of muscle excitation-contraction coupling.     

Intracellular sodium in mammalian muscle fibers after eccentric contractions.

The effect of eccentric contractions on intracellular Na(+) concentration ([Na(+)](i)) and its distribution were examined in isolated rat and mouse muscle fiber bundles. [Na(+)](i) was measured with either Na(+)-binding benzofuran isophthalate or sodium green. Ten isometric contractions had no significant effect on force (measured after 5 min of recovery) and caused no significant change in the resting [Na(+)](i) (7.2 +/- 0.5 mM). In contrast 10 eccentric contractions (40% stretch at 4 muscle lengths/s) reduced developed force at 100 Hz to 45 +/- 3% of control and increased [Na(+)](i) to 16.3 +/- 1.6 mM (n = 6; P < 0.001). The rise of [Na(+)](i) occurred over 1-2 min and showed only minimal recovery after 30 min. Confocal images of the distribution of [Na(+)](i) showed a spatially uniform distribution both at rest and after eccentric contractions. Gd(3+) (20 microM) had no effect on resting [Na(+)](i) or control tetanic force but prevented the rise of [Na(+)](i) and reduced the force deficit after eccentric damage. These data suggest that Na(+) entry after eccentric contractions may occur principally through stretch-sensitive channels.     

Dihydropyridine receptor-ryanodine receptor interactions in skeletal muscle excitation-contraction coupling

Much recent progress has been made in our understanding of the mechanism of sarcoplasmic reticulum Ca²⁺ release in skeletal muscle. Vertebrate skeletal muscle excitation-contraction (E-C) coupling is thought to occur by a mechanical coupling mechanism involving protein-protein interactions that lead to activation of the sarcoplasmic reticulum (SR) ryanodine receptor (RyR)/Ca²⁺ release channel by the voltage-sensing transverse (T–) tubule dihydropyridine receptor (DHPR)/Ca²⁺ channel. In a subsequent step, the released Ca²⁺ amplify SR Ca²⁺ release by activating release channels that are not linked to the DHPR. Experiments with mutant muscle cells have indicated that skeletal muscle specific DHPR and RyR isoforms are required for skeletal muscle E-C coupling. A direct functional and structural interaction between a DHPR-derived peptide and the RyR has been described. The interaction between the DHPR and RyR may be stabilized by other proteins such as triadin (a SR junctional protein) and modulated by phosphorylation of the DHPR.     

Electromechanical delay in human skeletal muscle under concentric and eccentric contractions

In contraction of skeletal muscle a delay exists between the onset of electrical activity and measurable tension. This delay in electromechanical coupling has been stated to be between 30 and 100 ms. Thus, in rapid movements it may be possible for electromyographic (EMG) activity to have terminated before force can be detected. This study was designed to determine the dependence of the EMG-tension delay upon selected initial conditions at the time of muscle activation. The right forearms of 14 subjects were passively oscillated by a motor-driven dynamometer through flexion-extension cycles of 135 deg at an angular velocity of approximately equal to 0.5 rad/s. Upon presentation of a visual stimulus the subjects maximally contracted the relaxed elbow flexors during flexion, extension, and under isometric conditions. The muscle length at the time of the stimulus was the same in all three conditions. An on-line computer monitoring surface EMG (Biceps and Brachioradialis) and force calculated the electromechanical delay. The mean value for the delay under eccentric condition, 49.5 ms, was significantly different (p less than 0.05) from the delays during isometric (53.9 ms) and concentric activity (55.5 ms). It is suggested that the time required to stretch the series elastic component (SEC) represents the major portion of the measured delay and that during eccentric muscle activity the SEC is in a more favorable condition for rapid force development.     

Thermodynamics of calmodulin binding to cardiac and skeletal muscle ryanodine receptor ion channels

The skeletal muscle (RyR1) and cardiac muscle (RyR2) ryanodine receptor calcium release channels contain a single, conserved calmodulin (CaM) binding domain, yet are differentially regulated by CaM. Here, we report that high-affinity [(35)S]CaM binding to RyR1 is driven by favorable enthalpic and entropic contributions at Ca(2+) concentrations from <0.01 to 100 microM. At 0.15 microM Ca(2+), [(35)S]CaM bound to RyR2 with decreased affinity and binding enthalpy compared with RyR1. The rates of [(35)S]CaM dissociation from RyR1 increased as the temperature was raised, whereas at 0.15 microM Ca(2+) the rate from RyR2 was little affected. The results suggest major differences in the energetics of CaM binding to and dissociation from RyR1 and RyR2.     

Cardiac ryanodine receptor is absent in type I slow skeletal muscle fibers: immunochemical and ryanodine binding studies

Cardiac ryanodine receptor was purified from canine ventricle as a single polypeptide of Mr 400,000 by a stepwise sucrose density gradient centrifugation and heparin-Sepharose CL-4B column chromatography. The [3H]ryanodine binding capacity (Bmax) was 60-fold enriched from cardiac microsomes without a change in affinity for [3H]ryanodine. The purity of the final preparation was determined to be greater than 95% by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using this purified preparation as an antigen, we produced six monoclonal antibodies which immunoprecipitated the cardiac ryanodine receptor. Three of these antibodies recognized the cardiac receptor on immunoblot analysis. In contrast, no protein in the microsomes isolated from Type I (slow) or Type II (fast) skeletal muscles was recognized by these antibodies. The [3H]ryanodine binding to cardiac and skeletal muscle microsomes was dependent on free Ca2+ concentration. In skeletal muscle microsomes, the [3H]ryanodine binding was remarkably enhanced by the addition of ATP or KCl and inhibited by high free Ca2+, whereas it was less sensitive to these agents in cardiac microsomes. All of these results clearly demonstrate that the cardiac ryanodine receptor is different from the skeletal muscle receptors and is not present even in Type I (slow) skeletal muscle fibers, in which cardiac isoforms of some of the muscle proteins are constitutively expressed.     

Complex interactions between skeletal muscle ryanodine receptor and dihydropyridine receptor proteins.

Evidence for functional interactions between the Ca2+ release channel in the skeletal muscle sarcoplasmic reticulum (the ryanodine receptor) and the L-type Ca2+ channel in the sarcolemma (the dihydropyridine receptor), leading to excitation-contraction coupling, is reviewed and experimental systems used to identify candidate sites of interaction are outlined.     

Indices of skeletal muscle damage and connective tissue breakdown following eccentric muscle contractions

 Indirect indices of exercise-induced human skeletal muscle damage and connective tissue breakdown were studied following a single bout of voluntary eccentric muscle contractions. Subjects (six female, two male), mean (SD) age 22 (2) years performed a bout of 50 maximum voluntary eccentric contractions of the knee extensors of a single leg. The eccentric exercise protocol induced muscle soreness (P < 0.05 Wilcoxon test), chronic force loss, and a decline in the 20:100 Hz percutaneous electrical myostimulation force ratio [P < 0.01, repeated measures analysis of variance (ANOVA)]. Serum creatine kinase (CK) and lactate dehydrogenase (LDH) activities were elevated (P < 0.01, repeated measures ANOVA) following the bout. The mean (SD) CK and LDH levels recorded 3 days post-exercise were 2815 (4144) IU · l^–1 and 375 (198) IU · l^–1, respectively. Serum alkaline phosphatase activity showed no changes throughout the study, and a non-significant increase (P = 0.058, repeated measures ANOVA) in pyridinoline was recorded following the bout. Urinary hydroxyproline (HP) and hydroxylysine (HL) excretion, expressed in terms of creatinine (Cr) concentration, increased after exercise (P < 0.05 and P < 0.01, respectively, repeated measures ANOVA). An increased HP:Cr was recorded 2 days post-exercise and HL:Cr was increased above baseline on days 2, 5, and 9 post-exercise. This indirect evidence of exercise-induced muscle damage suggests that myofibre disruption was caused by the eccentric muscle contractions. Elevated urine concentrations of indirect indices of collagen breakdown following eccentric muscle contractions suggests an increased breakdown of connective tissue, possibly due to a localised inflammatory response. Accepted: 9 October 1996     

Skeletal muscle collagen content in humans after high-force eccentric contractions.

The purpose of this study was to investigate the effects of high-force eccentric muscle contractions on collagen remodeling and on circulating levels of matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in humans. Nine volunteers [5 men and 4 women, mean age 23 (SD 4) yr] each performed a bout of 100 maximum voluntary eccentric contractions of the knee extensors. Muscle biopsies were taken before exercise and on days 4 and 22 afterward. Image analysis of stained tissue sections was used to quantify endomysial collagen staining intensity. Maximum voluntary contractile isometric force was recorded preexercise and on days 1, 2, 3, 4, 8, 11, and 14 postexercise. Venipuncture blood samples were also drawn on these days for measurement of serum creatine kinase activity and concentrations of MMP-9, TIMP-1, TIMP-2, and the MMP-2/TIMP-2 complex. Maximum voluntary contractile force declined by 39 +/- 23% (mean +/- SD) on day 2 postexercise and recovered thereafter. Serum creatine kinase activity peaked on day 4 postexercise (P < 0.01). Collagen type IV staining intensity increased significantly on day 22 postexercise to 126 +/- 29% (mean +/- SD) of preexercise values (P < 0.05). Serum MMP-9 levels increased on day 8 postexercise (P < 0.01), and serum TIMP-1 was also significantly elevated on days 1, 2, 3, 4, and 14 postexercise (P < 0.05). These results suggest that a single bout of eccentric muscle contractions results in remodeling of endomysial type IV collagen, possibly via the MMP pathway.     

Dihydropyridine effects on skeletal muscle fatigue.

1. The purpose of this investigation was to determine if alterations in extracellular calcium (Ca2+) influx by the dihydropyridine derivatives Bay K 8644 and nifedipine affected skeletal muscle fatigue. 2. Tetanic contractions (80 Hz, 100 msec) of frog sartorius muscles were evoked every sec for 3 min. Muscles were fatigued in normal Ringer's solution (NR), in NR containing 1 microM nifedipine of 10 microM Bay K 8644 or in low Ca2+ Ringer's. 3. In each case, the experimental conditions increased the rate and magnitude of fatigue. Rate constants of fatigue obtained during Bay K 8644, nifedipine and low Ca2+ conditions (-.0122 +/- .0016, -.0397 +/- 0022 and 0.0169 +/- .0064 sec-1, respectively) were significantly greater than NR (-.0104 +/- .0006 sec-1, p less than .05). In addition, tetanic forces developed at the end of the stimulation period under the experimental conditions (3.90 +/- 0.81, 1.21 +/- 1.40 and 2.04 +/- 1.10% of initial) were significantly less than NR (7.18 +/- 1.27%, p less than .05). 4. Caffeine contracture forces (10 mM) evoked immediately after stimulation were not significantly different between conditions. 5. These results suggest that alterations in sarcolemmal Ca2+ exchange has some influence on the fatigue process.     

Segmental muscle fiber lesions after repetitive eccentric contractions

Immunohistochemical and electron-microscopic techniques were used to analyze the extensor digitorum longus muscles of New Zealand White rabbits 1 h, 1 day, 3, 7, and 28 days after repetitive eccentric contractions. Loss of the cytoskeletal protein desmin was the earliest manifestation of injury. Apart from 1 h post-exercise, all desmin-negative fibers stained positively with antibody to plasma fibronectin, indicating loss of cellular integrity accompanying cytoskeletal disruption. Fiber sizes were significantly increased from 1–7 days after exercise. The large (hyaline) fibers found in histological sections after repetitive eccentric contractions resulted from segmental hypercontraction of the fiber. This phenomenon occurred proximally and distally to plasma membrane lesions of the muscle fiber and necrosis and manifested itself as very short sarcomere lengths. Thus, in serial sections, staining characteristics, sizes and shapes of one and the same fiber often varied dramatically. We conclude that the following sequence of events occurs: cytoskeletal disruptions, loss of myofibrillar registry, i.e., Z-disk streaming and A-band disorganization, and loss of cell integrity as manifested by intracellular plasma fibronectin stain, hypercontracted regions, and invasion of cells. When a fiber is disrupted, the remaining intact fibers apparently take up the tension put on the muscle and later fewer fibers are subjected to eccentric contractions.     

Effects of low-intensity training on dihydropyridine and ryanodine receptor content in skeletal muscle of mouse

To evaluate low-intensity exercise training induced changes in the expression of dihydropyridine (DHP) and ryanodine (Ry) receptors both mRNA and protein levels were determined by quantitative RT-PCR and immunoblot analysis from gastrocnemius (GAS) and rectus femoris (RF) muscles of mice subjected to a 15-week aerobic exercise program. The level of muscular work was assayed by changes in myosin heavy chain (MHC) content, myoglobin (Mb) expression and muscle size. The mRNA expression and optical density of DHP receptor increased significantly in GAS by 66.8 and 39.5%, respectively. The expression of Ry receptor, on the other hand, was not up-regulated. In RF, there was a significant increase of 38.4% in the mRNA expression of DHP receptor, although the protein level remained the same. No changes in Ry receptor expression was observed. The training resulted in a 1.58% increase in the amount of MHC IIa and a 2.34% decrease in that of IIb and IId in GAS. A significant 8.3% increase in the Mb content was observed. In RF, no significant changes in MHC or in Mb content were noted. Our results show that an evident increase in the mRNA and protein expression of DHP receptor was induced in GAS even by a relatively low-intensity exercise. Surprisingly, contrast to DHP receptor expression, no changes in Ry receptor mRNA, or protein levels were found, indicating more abundant demand for DHP receptor after increased muscle activity.     

Localization of calmodulin binding sites on the ryanodine receptor from skeletal muscle by electron microscopy.

Calmodulin (CaM) is a regulator of the calcium release channel (ryanodine receptor) of the sarcoplasmic reticulum of skeletal and cardiac muscle. The locations where CaM binds on the surface of the skeletal muscle ryanodine receptor were determined by electron microscopy. Wheat germ CaM was labeled specifically at Cys-27 with a maleimide derivative of a 1.4-nm-diameter gold cluster, and the gold-cluster-labeled CaM was bound to the purified ryanodine receptor. The complexes were imaged in the frozen-hydrated state by cryoelectron microscopy with no stains or fixatives present. In the micrographs, gold clusters were frequently observed near the corners of the square-shaped images of the ryanodine receptors. In some images, all four corners of the receptor were occupied by gold clusters. Image averaging allowed the site of CaM binding to be determined in two dimensions with an estimated precision of 4 nm. No changes were apparent in the quaternary structure of the ryanodine receptor upon binding CaM to the resolution attained, about 3 nm. Side views of the ryanodine receptor, in which the receptor is oriented approximately perpendicular to the much more frequent fourfold symmetric views, were occasionally observed, and showed that the CaM binding site is most likely on the surface of the receptor that faces the cytoplasm. We conclude that the CaM binding site is at least 10 nm from the transmembrane channel of the receptor and, consequently, that long-range conformational changes are involved in the modulation of the calcium channel activity of the receptor by CaM.     

A skeletal muscle ryanodine receptor interaction domain in triadin

Excitation-contraction coupling in skeletal muscle depends, in part, on a functional interaction between the ligand-gated ryanodine receptor (RyR1) and integral membrane protein Trisk 95, localized to the sarcoplasmic reticulum membrane. Various domains on Trisk 95 can associate with RyR1, yet the domain responsible for regulating RyR1 activity has remained elusive. We explored the hypothesis that a luminal Trisk 95 KEKE motif (residues 200-232), known to promote RyR1 binding, may also form the RyR1 activation domain. Peptides corresponding to Trisk 95 residues 200-232 or 200-231 bound to RyR1 and increased the single channel activity of RyR1 by 1.49±0.11-fold and 1.8±0.15-fold respectively, when added to its luminal side. A similar increase in [(3)H]ryanodine binding, which reflects open probability of the channels, was also observed. This RyR1 activation is similar to activation induced by full length Trisk 95. Circular dichroism showed that both peptides were intrinsically disordered, suggesting a defined secondary structure is not necessary to mediate RyR1 activation. These data for the first time demonstrate that Trisk 95's 200-231 region is responsible for RyR1 activation. Furthermore, it shows that no secondary structure is required to achieve this activation, the Trisk 95 residues themselves are critical for the Trisk 95-RyR1 interaction.     

Decreased contraction economy in mouse EDL muscle injured by eccentric contractions

Warren III, Gordon L., Jay H. Williams, Christopher W. Ward,Hideki Matoba, Christopher P. Ingalls, Karl M. Hermann, and R. B. Armstrong. Decreased contraction economy in mouse EDL muscleinjured by eccentric contractions. J. Appl.Physiol. 81(6): 2555-2564, 1996.The objective ofthis study was to find out whether basal and/or active energymetabolism are altered in isolated mouse extensor digitorum longusmuscle injured by eccentric (Ecc) contractions. Measurements of basalO₂ consumption and isometric tetanus O₂ recovery cost were madeat 25°C on muscles that had done either 10 Ecc, 10 isometric (Iso),or no contractions (No). In parallel experiments, rates of lactate andpyruvate production were measured to estimate the anaerobiccontribution. Basal O₂ consumptionwas unaffected by the type of protocol performed(P = 0.07). However, the tetanusO₂ cost per force-time integral was elevated by 30-36% for the Ecc protocol muscles over that forthe Iso and No protocol muscles. When including the increased lactateproduction by the Ecc protocol muscles, the total energetic cost perforce-time integral was 53% higher than that for the Iso protocolmuscles [2.35 ± 0.17 vs. 1.54 ± 0.18 µmolO₂/(N ^· m ^· s)].The decreased economy was attributed to two factors. First, in skinnedfibers isolated from the injured muscles, the ratio of maximalactomyosin adenosinetriphosphatase activity to force production was upby 37.5%, suggesting uncoupling of ATP hydrolysis from forceproduction. Second, increased reliance on anaerobic metabolism alongwith the fluorescent microscopic study of mitochondrial membranepotential and histochemical study of ATP synthase suggested anuncoupling of oxidative phosphorylation in the injured muscles.

     

Chicken skeletal muscle ryanodine receptor isoforms: ion channel properties.

To define the roles of the alpha- and beta-ryanodine receptor (RyR) (sarcoplasmic reticulum Ca2+ release channel) isoforms expressed in chicken skeletal muscles, we investigated the ion channel properties of these proteins in lipid bilayers. alpha- and beta RyRs embody Ca2+ channels with similar conductances (792, 453, and 118 pS for K+, Cs+ and Ca2+) and selectivities (PCa2+/PK+ = 7.4), but the two channels have different gating properties. alpha RyR channels switch between two gating modes, which differ in the extent they are activated by Ca2+ and ATP, and inactivated by Ca2+. Either mode can be assumed in a spontaneous and stable manner. In a low activity mode, alpha RyR channels exhibit brief openings (tau o = 0.14 ms) and are minimally activated by Ca2+ in the absence of ATP. In a high activity mode, openings are longer (tau o1-3 = 0.17, 0.51, and 1.27 ms), and the channels are activated by Ca2+ in the absence of ATP and are in general less sensitive to the inactivating effects of Ca2+. beta RyR channel openings are longer (tau 01-3 = 0.34, 1.56, and 3.31 ms) than those of alpha RyR channels in either mode. beta RyR channels are activated to a greater relative extent by Ca2+ than ATP and are inactivated by millimolar Ca2+ in the absence, but not the presence, of ATP. Both alpha- and beta RyR channels are activated by caffeine, inhibited by Mg2+ and ruthenium red, inactivated by voltage (cytoplasmic side positive), and modified to a long-lived substate by ryanodine, but only alpha RyR channels are activated by perchlorate anions. The differences in gating and responses to channel modifiers may give the alpha- and beta RyRs distinct roles in muscle activation.     

High-resolution structure of the membrane-embedded skeletal muscle ryanodine receptor

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Response to caffeine and ryanodine receptor isoforms in mouse skeletal muscles

The response to caffeine was studied in mouse muscles[diaphragm, soleus, and extensor digitorum longus (EDL)] withdifferent ryanodine receptor isoform (RyR1, RyR3) composition and insingle permeabilized muscle fibers dissected from diaphragm ofwild-type (WT) and RyR3-deficient (RyR3/) mice at 1, 15, 30, and 60 postnatal days (PND). The caffeine response decreased duringdevelopment, and, in adult mice, was greater in diaphragm, lower inEDL, and intermediate in soleus. This suggests a direct relationbetween response to caffeine and RyR3 expression. The lack of RyR3reduced caffeine response in young, but not in adult mice, and did not abolish the age-dependent variation and the intermuscle differences. Indiaphragm single fibers, the response to caffeine increased duringdevelopment and was reduced in fibers lacking RyR3 both at 15 and 60 PND. A population of fibers highly responsive to caffeine was presentin adult WT and disappeared in RyR3/. The results confirm thecontribution of RyR3 to calcium release for contractile response andclarify the contribution of RyR3 to developmental changes andintermuscle differences.