Changes in actin structural transitions associated with oxidative inhibition of muscle contraction

We have used transient phosphorescence anisotropy (TPA) to detect changes in actin structural dynamics associated with oxidative inhibition of muscle contraction. Contractility of skinned rabbit psoas muscle fibers was inhibited by treatment with 50 mM H 2O 2, which induced oxidative modifications in the myosin head and in actin, as previously reported. Using proteins purified from oxidized and unoxidized muscle, we used TPA to measure the effects of weakly (+ATP) and strongly (no ATP) bound myosin heads (S1) on the microsecond dynamics of actin labeled at Cys374 with erythrosine iodoacetamide. Oxidative modification of S1 had no effect on actin dynamics in the absence of ATP (strong binding complex), but restricted the dynamics in the presence of ATP (weakly bound complex). In contrast, oxidative modification of actin did not have a significant effect on the weak-to-strong transitions. Thus, we concluded that (1) the effects of oxidation on the dynamics of actin in the actomyosin complex are predominantly determined by oxidation-induced changes in S1, and (2) changes in weak-to-strong structural transitions in actin and myosin are coupled to each other and are associated with oxidative inhibition of muscle contractility.     

Oxidant activity in skeletal muscle fibers is influenced by temperature, CO2 level, and muscle-derived nitric oxide

Free radicals are produced continuously by skeletal muscle fibers. Extracellular release of reactive oxygen species (ROS) and nitric oxide (NO) derivatives has been demonstrated, but little is known about intracellular oxidant regulation. We used a fluorescent oxidant probe, 2',7'-dichlorofluorescin (DCFH), to assess net oxidant activity in passive muscle fiber bundles isolated from mouse diaphragm and studied in vitro. We tested the following three hypotheses. 1) Net oxidant activity is decreased by muscle cooling. 2) CO(2) exposure depresses intracellular oxidant activity. 3) Muscle-derived ROS and NO both contribute to overall oxidant activity. Our results indicate that DCFH oxidation was diminished by cooling muscle fibers from 37 degrees C to 23 degrees C (P < 0.001). The rate of DCFH oxidation correlated positively with CO(2) exposure (0-10%; P < 0.05) and negatively with concurrent changes in pH (7.0-8.5; P < 0.05). Separate exposures to anti-ROS enzymes (superoxide dismutase, 1 kU/ml; catalase, 1 kU/ml), a glutathione peroxidase mimetic (ebselen, 30 microM), NO synthase inhibitors (N(omega)-nitro-l-arginine methyl ester, 1 mM; N(omega)-monomethyl-l-arginine, 1 mM), or an NO scavenger (hemoglobin, 1 microM) each inhibited DCFH oxidation (P < 0.05). Oxidation was increased by hydrogen peroxide, 100 microM, an NO donor (NOC-22, 400 microM), or the substrate for NO synthase (l-arginine, 5 mM). We conclude that net oxidant activity in resting muscle fibers is 1) decreased at subphysiological temperatures, 2) increased by CO(2) exposure, and 3) influenced by muscle-derived ROS and NO derivatives to similar degrees.     

Butanedione monoxime suppresses contraction and ATPase activity of rabbit skeletal muscle

The effects of 2,3-butanedione 2-monoxime (BDM) on mechanical responses of glycerinated fibers and the ATPase activity of heavy meromyosin (HMM) and myofibrils have been studied using rabbit skeletal muscle. The mechanical responses and the ATPase activity were measured in similar conditions (ionic strength 0.06-0.2 M, 0.4-4 mM MgATP, 0-20 mM BDM, 2-20 degrees C and pH 7.0). BDM reversibly reduced the isometric tension, shortening speed, and instantaneous stiffness of the fibers. BDM also inhibited myofibrillar and HMM ATPase activities. The inhibitory effect on the relative ATPase activity of HMM was not influenced by the addition of actin or troponin-tropomyosin-actin. High temperature and low ionic strength weakened BDM's suppression of contraction of the fibers and the ATPase activity of contracting myofibrils, but not of the HMM, acto-HMM and relaxed myofibrillar ATPase activity. The size of the initial phosphate burst at 20 degrees C was independent of the concentration of BDM. These results suggest that the suppression of contraction of muscle fibers is due mainly to direct action of BDM on the myosin molecules.     

Catalytic cooperativity induced by SH1 labeling of myosin filaments

Modifications of SH1 groups on isolated myosin subfragment 1 (S-1) and myosin in muscle fibers affect differently the acto-S-1 ATPase and the fiber properties. Consistent with the findings of earlier work on fibers, the modification of SH1 groups in relaxed myofibrils with phenylmaleimide caused a loss of their shortening. This loss paralleled the decrease in the Vmax of extracted myosin but was not linear with the extent of SH1 labeling. Strikingly, the decrease in Vmax of S-1 prepared from the modified myofibrils was directly proportional to the extent of SH1 labeling. The specificity of SH1 labeling in myofibrils was verified by ATPase activities, thiol titrations, radiolabeling experiments, and comparisons to myosin labeled on SH1 in solution. To test for intermolecular interactions in the myosin filaments and their contribution to the differences between S-1 and myosin, the catalytic properties of copolymers of myosin were examined. Copolymers of myosin and rod minifilaments were formed in 5 mM citrate-Tris (pH 8.0) buffer, and their homogeneity was verified by sedimentation velocity analysis. The inhibition of actomyosin ATPase by rod particles was related to the decrease in the Km value. When rod particles were replaced in these minifilaments by SH1-modified myosin, the ATPase of the copolymers was increased over that of the combined ATPases of the individual filaments. The actomyosin ATP turnover rates on the unmodified heads were increased severalfold by the modified heads.(ABSTRACT TRUNCATED AT 250 WORDS)     

Myosin light chain phosphorylation inhibits muscle fiber shortening velocity in the presence of vanadate

We have shown that myosin light chain phosphorylation inhibits fiber shortening velocity at high temperatures, 30 degrees C, in the presence of the phosphate analog vanadate. Vanadate inhibits tension by reversing the transition to force-generating states, thus mimicking a prepower stroke state. We have previously shown that at low temperatures vanadate also inhibits velocity, but at high temperatures it does not, with an abrupt transition in inhibition occurring near 25 degrees C (E. Pate, G. Wilson, M. Bhimani, and R. Cooke. Biophys J 66: 1554-1562, 1994). Here we show that for fibers activated in the presence of 0.5 mM vanadate, at 30 degrees C, shortening velocity is not inhibited in dephosphorylated fibers but is inhibited by 37 +/- 10% in fibers with phosphorylated myosin light chains. There is no effect of phosphorylation on fiber velocity in the presence of vanadate at 10 degrees C. The K(m) for ATP, defined by the maximum velocity of fibers partially inhibited by vanadate at 30 degrees C, is 20 +/- 4 microM for phosphorylated fibers and 192 +/- 40 microM for dephosphorylated fibers, showing that phosphorylation also affects the binding of ATP. Fiber stiffness is not affected by phosphorylation. Inhibition of velocity by phosphorylation at 30 degrees C depends on the phosphate analog, with approximately 12% inhibition in fibers activated in the presence of 5 mM BeF(3) and no inhibition in the presence of 0.25 mM AlF(4). Our results show that myosin phosphorylation can inhibit shortening velocity in fibers with large populations of myosin heads trapped in prepower stroke states, such as occurs during muscle fatigue.     

Effect of inorganic phosphate on the Ca2+ sensitivity in skinned Taenia coli smooth muscle fibers. Comparison of tension, ATPase activity, and phosphorylation of the regulatory myosin light chains.

Inorganic phosphate (Pi) decreases maximal tension in contracted skeletal and heart muscle fibers. We investigated the effects of 10 mM Pi on the force-calcium relationship in Triton X-100-skinned Taenia coli smooth muscle fibers. Isometric force measurements show that the calcium sensitivity of the force depends on the phosphate concentration. Furthermore 10 mM Pi relaxes the fibers more at intermediate than at high calcium ion concentrations: At pCa 4.5 tension decreases in the presence of 10 mM Pi by approximately 12% but it decreases 70% at pCa 6.17. Removal of phosphate partially reverses the relaxation. Simultaneous determination of actomyosin ATPase activity and force (Güth, K., and J. Junge, 1982, Nature (Lond.), 300:775-776) shows that the ATPase activity does not correlate with the changes in force. In the presence of Pi, tension decreases more than the ATPase activity. The level of phosphorylation of the 20,000-D regulatory myosin light chain is not changed in the presence or absence of 10 mM Pi. The results are discussed in terms of slowly or noncycling myosin crossbridges formed at lower calcium concentrations, which contribute to the force development but not to the ATPase activity. These crossbridges are considered to be dissociated in the presence of phosphate.     

Effects of hindlimb unweighting and aging on rat semimembranosus muscle and myosin.

We tested the hypothesis that lower specific force (force/cross-sectional area) generated by type II fibers from hindlimb-unweighted rats resulted from structural changes in myosin (i.e., a change in the ratio of myosin cross bridges in the weak- and strong-binding state during contraction). In addition, we determined whether those changes were age dependent. Permeabilized semimembranosus muscle fibers from young adult and aged rats, some of which were hindlimb unweighted for 3 wk, were studied for Ca(2+)-activated force generation and maximal unloaded shortening velocity. Fibers were also spin labeled specifically at myosin Cys707 to assess the structural distribution of myosin during maximal isometric contraction using electron paramagnetic resonance spectroscopy. Myosin heavy chain isoform (MHC) expression and the ratio of MHC to actin were evaluated in each fiber. Fibers from the unweighted rats generated 34% less specific force than fibers from weight-bearing rats (P < 0.001), independent of age. Electron paramagnetic resonance analyses showed that the fraction of myosin heads in the strong-binding structural state during contraction was 11% lower in fibers from the unweighted rats (P = 0.019), independent of age. More fibers from unweighted rats coexpressed MHC IIB-IIX compared with fibers from weight-bearing rats (P = 0.049). Unweighting induced a slowing of maximal unloaded shortening velocity and an increase in the ratio of MHC to actin in fibers from young rats only. These data indicate that altered myosin structural distribution during contraction and a preferential loss of actin contribute to unweighting-induced muscle weakness. Furthermore, the age of the rat has an influence on some parameters of changes in muscle contractility that are induced by unweighting.     

Inhibitors arrest myofibrillogenesis in skeletal muscle cells at early stages of assembly

A three-step model for myofibrillogenesis has been proposed for the formation of myofibrils [Rhee et al., 1994: Cell Motil. Cytoskeleton 28:1-24; Sanger et al., 2002: Adv. Exp. Med. 481:89-105]: premyofibril to nascent myofibril to mature myofibril. We have found two chemically related inhibitors that will arrest development at both the first and second step. Cultured quail embryonic skeletal myoblasts were treated with ethyl methane sulfonate (EMS) or 2-aminoethyl-methanesulfonate (MTSEA+). When the myoblasts fused in the presence of either of these compounds, myosheets rather than myotubes formed. Treated cells were fixed and immunostained against multiple proteins commonly found in muscle cells. Protein expression and localization throughout the myosheet were similar to that of developing myotube tips. Cells treated with high concentrations of EMS (10 mM) stained for non-muscle myosin II, sarcomeric alpha-actinin, and tropomyosin. No zeugmatin (Z-band region of titin) or muscle myosin II antibody staining was detected in fibers in this treatment group. These fibers are comparable to premyofibrils in control myotubes. At lower concentrations of EMS (7.5 to 5 mM), fibers that formed stained for muscle myosin II and titin as well as for non-muscle myosin IIB, sarcomeric alpha-actinin, and tropomyosin. Muscle myosin II was in an unbanded pattern. These fibers are comparable to nascent myofibrils observed during normal myofibrillogenesis. Similar effects to those obtained by treating cells with EMS were obtained when we treated cultured cells with MTSEA+ (5 mM) and stained them with sarcomeric alpha-actinin. MTSEA+ is chemically related to EMS, and is a well-known inhibitor of ryanodine receptors in skeletal muscle cells. Some abnormalities such as nemaline-like rods and other protein aggregates also appear within the myosheet during EMS and MTSEA+ treatment. Removal of these two inhibitors of myofibrillogenesis allows the premyofibrils and nascent myofibrils to form mature myofibrils.     

Effect of actomyosin contractility on focal contacts of myofibroblasts and structure of stress fibers

Myofibroblasts from rat lung were cultivated. These cells in addition to beta- and gamma-cytoplasmic actins, expressed alpha-smooth muscle actin (alpha-SMA) and formed a system of "supermature" focal contacts, which were connected with thick stress-fibers expressing alpha-SMA and myosin II. Reduction of actin-myison contractility by inhibitors BDM and ML-7 lead to stress fiber reorganization, e.g., decrease in their thickness, a selective disappearance of alpha-SMA expression and myosin translocation from bundles to the cytoplasm. Using immunofluorescence, interference-reflection microscopy and morphometry, we have demonstrated that an inhibition of actin-myosin contractility also leads to dispersion of myofibroblastic focal contacts. Phase-contrast and DIC video-enhanced microscopy of live cells showed morphological reorganization at the leading edge after inhibitory treatment. Thus, actin-myosin contractility controls the structure of "supermature" focal contacts of myofibroblasts and alpha-SMA expression in stress fibers.     

Effects of pH on contraction of rabbit fast and slow skeletal muscle fibers.

We have investigated (a) effects of varying proton concentration on force and shortening velocity of glycerinated muscle fibers, (b) differences between these effects on fibers from psoas (fast) and soleus (slow) muscles, possibly due to differences in the actomyosin ATPase kinetic cycles, and (c) whether changes in intracellular pH explain altered contractility typically associated with prolonged excitation of fast, glycolytic muscle. The pH range was chosen to cover the physiological pH range (6.0-7.5) as well as pH 8.0, which has often been used for in vitro measurements of myosin ATPase activity. Steady-state isometric force increased monotonically (by about threefold) as pH was increased from pH 6.0; force in soleus (slow) fibers was less affected by pH than in psoas (fast) fibers. For both fiber types, the velocity of unloaded shortening was maximum near resting intracellular pH in vivo and was decreased at acid pH (by about one-half). At pH 6.0, force increased when the pH buffer concentration was decreased from 100 mM, as predicted by inadequate pH buffering and pH heterogeneity in the fiber. This heterogeneity was modeled by net proton consumption within the fiber, due to production by the actomyosin ATPase coupled to consumption by the creatine kinase reaction, with replenishment by diffusion of protons in equilibrium with a mobile buffer. Lactate anion had little mechanical effect. Inorganic phosphate (15 mM total) had an additive effect of depressing force that was similar at pH 7.1 and 6.0. By directly affecting the actomyosin interaction, decreased pH is at least partly responsible for the observed decreases in force and velocity in stimulated muscle with sufficient glycolytic capacity to decrease pH.     

Connectin filaments in stretched skinned fibers of frog skeletal muscle

Indirect immunofluorescence microscopy of highly stretched skinned frog semi-tendinous muscle fibers revealed that connectin, an elastic protein of muscle, is located in the gap between actin and myosin filaments and also in the region of myosin filaments except in their centers. Electron microscopic observations showed that there were easily recognizable filaments extending from the myosin filaments to the I band region and to Z lines in the myofibrils treated with antiserum against connectin. In thin sections prepared with tannic acid, very thin filaments connected myosin filaments to actin filaments. These filaments were also observed in myofibrils extracted with a modified Hasselbach-Schneider solution (0.6 M KCl, 0.1 M phosphate buffer, pH 6.5, 2 mM ATP, 2 mM MgCl2, and 1 mM EGTA) and with 0.6 M Kl. SDS PAGE revealed that connectin (also called titin) remained in extracted myofibrils. We suggest that connectin filaments play an important role in the generation of tension upon passive stretch. A scheme of the cytoskeletal structure of myofibrils of vertebrate skeletal muscle is presented on the basis of our present information of connectin and intermediate filaments.     

Inhibition of muscle force by vanadate.

Vanadate (Vi), an analogue of inorganic phosphate (Pi), is known to bind tightly with a long half life to the myosin MgATPase site, producing a complex which inhibits force. Both of these ligands bind to an actin.myosin.ADP state that follows the release of Pi in the enzymatic cycle, and their effects on muscle fibers and proteins in solution provide information on the properties of this state. The inhibition of active force generation began to occur at a [Vi] of 5 microM and was 90% complete at a [Vi] of 1 mM. Hill plots of the inhibition of force by Vi approximated that expected for a simple binding isotherm. Similar plots were obtained at both 25 degrees C and 5 degrees C. A simple binding isotherm is not expected to occur in a muscle fiber where steric constraints imposed by the intact filaments should introduce more complexity into the energetics of ligand binding. The inhibition of MgATPase activity for acto-subfragment-1 to 50% of controls occurred at a [Vi] which was only 20-fold higher than that required to inhibit force generation in fibers to the same level. Some models of actomyosin interactions would predict that the range of [Vi] required for complete force inhibition in fibers and the difference in the [Vi] required for inhibition in fibers and of myosin in solution would both be much larger.     

Micromethods in single muscle fibers. 1. Determination of catalase and superoxide dismutase

Methods have been developed for the measurements of catalase and superoxide dismutase (SOD) in single, isolated muscle fibers. These fibers are also classified according to fiber type. Catalase is determined using a fluorescent method for the measurement of hydrogen peroxide consumed. SOD measurements are carried out using a modification of established techniques whereby the inhibition of oxidation of epinephrine by SOD is assayed fluorometrically. Both enzymes may be determined in submicrogram samples of dried muscle. This approach avoids the complication of the inclusion of nonmuscle tissue with varying enzymatic activities which is frequently experienced when using homogenates of muscle, particularly diseased muscle. In addition, these techniques can be used to determine the inherent variation in SOD and catalase activities within individual fibers of the same fiber type. The Km and Vmax for catalase, determined using homogenates of human muscle, were found to be 12 mM and 1.45 mumol/min/mg dry wt, respectively. Catalase of muscle was inhibited 50% by 2 microM sodium azide. Mn-SOD contributes less than one-fifth of the total SOD activity. Therefore the activity is largely due to the Cu-Zn form of SOD. These methods are applicable to a wide variety of tissues.     

Effects of acute and chronic ethanol on cardiac contractile protein ATPase activity of Syrian hamsters

Ethanol consumption is known to affect cardiac and skeletal muscle. In vivo experiments on cardiac muscle showed that ethanol affects cardiac contractility and Vmax, suggesting that contractile proteins of the myocardium were affected by ethanol. Therefore, experiments were carried out to examine the effects of ethanol on the cardiac contractile protein ATPase activities. Cardiac myofibrils isolated from ethanol-fed hamsters showed a significant decrease in myofibrillar ATPase activities between pCa 6 and 4. On the other hand, addition of ethanol (0.1%) in vitro to cardiac myofibrils from control hamster had no significant effect on the ATPase activities, suggesting that hamsters need to be exposed for longer periods of time to induce demonstratable changes in the contractile protein ATPase activity. Actin-activated myosin ATPase activities were significantly lower in myofibrils from ethanol-fed hamsters at 1:1 and 1:2 ratios of myosin to actin. These investigations revealed that chronic (4 weeks) exposure of hamsters to ethanol reduced cardiac contractile protein ATPase activity, which may help explain impaired cardiac function in chronic alcoholics.     

Effect of phosphate and acidosis on the calcium sensitivity of the cardiac myofibrils

The treatment of the bundles of rat myocardial fibers with ethyleneglycol-bis(beta-aminoethyl ether)-N,N-tetraacetate (EGTA) made the sarcolemma permeable for ions and small molecules. At the incubation medium pH 7.0 the EGTA-treated fibers developed a half-maximal tension at pCa 5.4, and the maximal tension at pCa 4.8. Inorganic phosphate (10 mM) reduced the maximal tension by 18 +/- 3% and decreased the calcium sensitivity of the myofibrils so that there was a shift of the pCa/tension curve by 0.3 unit to the right. Acidosis (pH 6.6) also decreased significantly the calcium sensitivity, while the presence of 10 mM phosphate produced additional depression of the calcium sensitivity. It is concluded that phosphate accumulation by the ischemic myocardium combined with acidosis may depress the contractility not only due to depletion of the free calcium concentration in the myoplasm but also as a result of the reduced calcium sensitivity of myofibrils.     

Decreased specific force and power production of muscle fibers from myostatin-deficient mice are associated with a suppression of protein degradation

Myostatin (MSTN) is a member of the transforming growth factor-β superfamily of cytokines and is a negative regulator of skeletal muscle mass. Compared with MSTN(+/+) mice, the extensor digitorum longus muscles of MSTN(-/-) mice exhibit hypertrophy, hyperplasia, and greater maximum isometric force production (F(o)), but decreased specific maximum isometric force (sF(o); F(o) normalized by muscle cross-sectional area). The reason for the reduction in sF(o) was not known. Studies in myotubes indicate that inhibiting myostatin may increase muscle mass by decreasing the expression of the E3 ubiquitin ligase atrogin-1, which could impact the force-generating capacity and size of muscle fibers. To gain a greater understanding of the influence of myostatin on muscle contractility, we determined the impact of myostatin deficiency on the contractility of permeabilized muscle fibers and on the levels of atrogin-1 and ubiquitinated myosin heavy chain in whole muscle. We hypothesized that single fibers from MSTN(-/-) mice have a greater F(o), but no difference in sF(o), and a decrease in atrogin-1 and ubiquitin-tagged myosin heavy chain levels. The results indicated that fibers from MSTN(-/-) mice have a greater cross-sectional area, but do not have a greater F(o) and have a sF(o) that is significantly lower than fibers from MSTN(+/+) mice. The extensor digitorum longus muscles from MSTN(-/-) mice also have reduced levels of atrogin-1 and ubiquitinated myosin heavy chain. These findings suggest that myostatin inhibition in otherwise healthy muscle increases the size of muscle fibers and decreases atrogin-1 levels, but does not increase the force production of individual muscle fibers.     

Mg-ATPase and CA2+ activated myosin ATPase activity in ventricular myofibrils from non-failing and diseased human hearts – effects of calcium sensitizing agents MCI-154, DPI 201–106, and caffeine

We investigated the effects of two purported calcium sensitizing agents, MCI-154 and DPI 201–106, and a known calcium sensitizer caffeine on Mg-ATPase (myofibrillar ATPase) and myosin ATPase activity of left ventricular myofibrils isolated from non-failing, idiopathic (IDCM) and ischemic cardiomyopathic (ISCM) human hearts (i.e. failing hearts). The myofibrillar ATPase activity of non-failing myofibrils was higher than that of diseased myofibrils. MCI-154 increased myofibrillar ATPase Ca²⁺ sensitivity in myofibrils from non-failing and failing human hearts. Effects of caffeine similarly increased Ca²⁺ sensitivity. Effects of DPI 201–106 were, however, different. Only at the 10^–6 M concentration was a significant increase in myofibrillar ATPase calcium sensitivity seen in myofibrils from non-failing human hearts. In contrast, in myofibrils from failing hearts, DPI 201–106 caused a concentration-dependent increase in myofibrillar ATPase Ca²⁺ sensitivity. Myosin ATPase activity in failing myocardium was also decreased. In the presence of MCI-154, myosin ATPase activity increased by 11, 19, and 24% for non-failing, IDCM, and ISCM hearts, respectively. DPI 201–106 caused an increase in the enzymatic activity of less than 5% for all preparations, and caffeine induced an increase of 4, 11, and 10% in non-failing, IDCM and ISCM hearts, respectively. The mechanism of restoring the myofibrillar Ca²⁺ sensitivity and myosin enzymatic activity in diseased human hearts is most likely due to enhancement of the Ca²⁺ activation of the contractile apparatus induced by these agents. We propose that myosin light chain-related regulation may play a complementary role to the troponin-related regulation of myocardial contractility.     

Probing the Regulation of Contractility in Cardiac and Smooth Muscle Skinned Fibers with Synthetic Peptides

Methods for using synthetic peptides to specifically probe the molecular mechanisms for calcium-dependent regulation of contraction in cardiac and smooth permeabilized (or skinned) muscle are described. As examples of the use of these tools, the role of troponin in modulating the cardiac crossbridge cycle and the regulatory action of myosin light chain kinase (MLCK) in smooth muscle in Triton X-100-extracted muscle preparations have been targeted. These "skinned" fibers are functional in terms of contractility but permit precise control of aspects of the "cytoplasmic" environment around the myofilaments, such as calcium and substrate concentration. They also permit the diffusion of peptides into the ′intracellular" compartment. These include peptides derived from the common actin-binding, troponin C-binding sequence of troponin I (the so-called inhibitory sequence, Tnl 104–115) and the calmodulin-binding sequence of MLCK (also known as RS20). The effects of these peptides were monitored in terms of changes in isometric tension and expressed as changes in calcium or calmodulin sensitivity. The calmodulin-binding peptide reduced force at a fixed calcium concentration, indicating decreased calcium sensitivity. This effect was associated with a moderate decrease in myosin light chain phosphorylation and could be reversed with increased calmodulin concentration. We interpret this latter observation to mean that underlying the change in apparent calcium sensitivity is a change in the sensitivity of MLCK to calmodulin. As previously reported, the troponin I-based peptide desensitizes skinned cardiac muscle with respect to calcium by inhibiting the actin activation of the crossbridge cycle. We also discuss the results of recent experiments in which this peptide was used in conjunction with a calcium-sensitizing compound, EMD 53998. These results implicate the phosphate release step as the most likely regulatory step in the crossbridge cycle affected by the peptide and, by extension, troponin I. Peptide studies such as these have provided useful specific insights into the highly complex and multivariable regulatory systems of contraction.     

Synthesis and properties of a conformationally restricted spin-labeled analog of ATP and its interaction with myosin and skeletal muscle.

The synthesis is described of a spin-labeled analog of ATP, 2',3'-O-(1-oxy-2,2,6,6-tetramethyl-4-piperidylidene)adenosine 5'-triphosphate (SL-ATP). The spin-label moiety is attached by two bonds to the ribose ring as a spiroketal and hence has restricted conformational mobility relative to the ribose moiety of ATP. The synthesis proceeds via an acid-catalyzed addition of adenosine 5'-monophosphate to 1-acetoxy-4-methoxy-2,2,6,6-tetramethyl-1,2,5,6-tetrahydropyridine in acetonitrile. The spiroketal product is pyrophosphorylated, and alkaline hydrolysis with concomitant aerial oxidation gives the required product. The spin-labeled moiety probably takes up two rapidly interconverting conformations with respect to the ribose ring on the basis of the 1H NMR spectra of its precursors and related uridine derivatives [Alessi et al. (1991) J. Chem. Soc., Perkin Trans.1,2243-2247]. SL-ATP is a substrate for myosin and actomyosin with similar kinetic parameters to ATP during triphosphatase activity. SL-ATP supports muscle contraction and permits relaxation of permeabilized rabbit skeletal muscle fibers. SL-ADP is a substrate for yeast 3-phosphoglycerate kinase, thus permitting regeneration of SL-ATP from SL-ADP within muscle fibers. Electron paramagnetic resonance (EPR) studies of SL-ADP bound to myosin filaments and to myofibrils show a degree of nanosecond motion independent of that of the protein, which may be due to conformational flexibility of the ribose moiety of ATP bound to myosin's active site. This nanosecond motion is more restricted in myofibrils than in myosin filaments, suggesting that the binding of actin affects the ribose binding site in myosin. EPR studies on SL-ADP bound to rigor cross-bridges in muscle fiber bundles showed the nucleotide to be highly oriented with respect to the fiber axis.     

The effects of ADP and phosphate on the contraction of muscle fibers.

The products of MgATP hydrolysis bind to the nucleotide site of myosin and thus may be expected to inhibit the contraction of muscle fibers. We measured the effects of phosphate and MgADP on the isometric tensions and isotonic contraction velocities of glycerinated rabbit psoas muscle at 10 degrees C. Addition of phosphate decreased isometric force but did not affect the maximum velocity of shortening. To characterize the effects of ADP on fiber contractions, force-velocity curves were measured for fibers bathed in media containing various concentrations of MgATP (1.5-4 mM) and various concentrations of MgADP (1-4 mM). As the [MgADP]/[MgATP] ratio in the fiber increases, the maximum velocity achieved by the fiber decreases while the isometric tension increases. The inhibition of fiber velocities and the potentiation of fiber tension by MgADP is not altered by the presence of 12 mM phosphate. The concentration of both MgADP and MgATP within the fiber was calculated from the diffusion coefficient for nucleotides within the fiber, and the rate of MgADP production within the fiber. Using the calculated values for the nucleotide concentration inside the fiber, observed values of the maximum contraction velocity could be described, within experimental accuracy, by a model in which MgADP competed with MgATP and inhibited fiber velocity with an effective Ki of 0.2-0.3 mM. The average MgADP level generated by the fiber ATPase activity within the fiber was approximately 0.9 mM. In fatigued fibers MgADP and phosphate levels are known to be elevated, and tension and the maximum velocity of contraction are depressed. The results obtained here suggest that levels of MgADP in fatigued fibers play no role in these decreases in function, but the elevation of both phosphate and H+ is sufficient to account for much of the decrease in tension.