Equatorial x-ray intensities and isometric force levels in frog sartorius muscle.

Isometric force levels, ranging between 0 and 100% of maximal force P₀ at 2 to 3 °C, were elicited in frog sartorius muscle by means of rapidly cooling a Ringer solution containing 1·25 to 2·0 mm-caffeine. Equatorial X-ray diffraction patterns were obtained in the resting state and during contraction. The ratio of the intensities $\text{I}{}_{11}\text{I}{}_{10}$ increased with force almost linearly, with a slight upward curvature. The individual intensities for the contracting state were normalized relative to both the intensity of the undiffracted beam and the intensity of each reflection in the resting state. These normalized intensities were found to vary in a reciprocal way: I₁₀ decreased while I₁₁ increased throughout the range of forces studied.The gradual change in $\text{I}{}_{11}\text{I}{}_{10}$ with force level indicates that this ratio is a sensitive measure of the number of cross-bridges in the isometric state. A two-state model in which myosin projections are either in a resting or attached state and in which force is proportional to the fraction of projections in the attached state was applied to the experimental data of the individual reflections. I₁₀ deviates from this model in a way that suggests that formation of the first few cross-bridges may decrease the regularity of the remaining unattached myosin projections.     

Influence of temperature upon contractile activation and isometric force production in mechanically skinned muscle fibers of the frog

Increasing temperature (4-22 degrees C) increases the Ca2+ concentration required for activation of mechanically skinned frog muscle fibers. The pCa required for 50% maximal force (pCa50) was inversely proportional to absolute temperature. Assuming that relative force is directly related to fractional occupancy of the Ca2+-binding sites on troponin that regulate force, the shift was consistent with a Gibbs free energy change of binding (delta G) of about -7.8 kcal/mol. This is close to the delta G for Ca2+ binding to the calcium-specific sites on troponin C reported by others. Decreasing Mg2+ from 1 mM to 60 microM shifts the force-pCa curves at either 4 or 22 degrees C to higher pCa, but the shift of pCa50 with temperature over this range (0.4 log units) was the same at low and high Mg2+. Maximal force increased with temperature for the entire range 4-22 degrees C with a Q10 of 1.41, and over the restricted range 4-15 degrees C with a Q10 of 1.20. From the dual effects of temperature on Ca2+ activation and maximal force, one would expect that force would respond differently to temperature change at high or low Ca2+. At high Ca2+, a temperature increase will lead to an increased force. However, at low to intermediate Ca2+ levels (below the intersection of the force-pCa curves for the initial and final temperatures), steady state force should decrease with increasing temperature. The inverse responses should occur with a decrease in temperature. These responses are observed when temperature is changed by rapid solution exchange.     

Chemical energetics of force development, force maintenance, and relaxation in mammalian smooth muscle

High-energy phosphate utilization (delta approximately P) associated with force development, force maintenance, and relaxation has been determined during single isometric tetani in the rabbit taenia coli. ATP resynthesis from glycolysis and respiration was stopped without deleterious effects on the muscle. At 18 degrees C and a muscle length of 95% l0, the resting rate of energy utilization is 1.8 +/- 0.2 nmol/g . s-1, or 0.85 +/- 0.2 mmol approximately P/mol of total creatine (Ct) . s-1, where Ct = 2.7 mumol/g wet wt. During the initial 25 s of stimulation when force is developed, the average rate of delta approximately P was -8.2 +/- 0.8 mmol/mol Ct . s-1, some four times greater than during the subsequent 35 s of force maintenance, when the rate was -2.0 +/- 0.6 mmol approximately P/mol Ct . s-1. The energy cost of force redevelopment (0 to 95% P0) after a quick release from the peak of a tetanus is very low compared with the initial force development. Therefore, the high rate of energy utilization during force development is not due only to internal work done against the series elasticity nor to any high rate of cross-bridge cycling inherently associated with force development. The high economy of force maintenance compared with other muscle types is undoubtedly due to a slower cross-bridge cycle time. The energy utilization during 45 s of relaxation was not statistically significant, and integral of Pdt/delta approximately P was higher during relaxation than during force maintenance in the stimulated muscle.     

Activation of phosphorylase in frog muscle as determined by contractile activity

The state of activation of phosphorylation in muscle has been reinvestigated by combining the extraction procedures of Danforth, Helmreich, and Cori with the low-temperature techniques of this laboratory. In resting frog muscle, the phosphorylase-alpha content is usually below detectability. Upon contractile activity in series of twitches, activation of phosphorylase beta to alpha took place, without activation of phosphorylase beta kinase as defined by the assay procedure. Two different experimental designs were used to examine the relation between phosphorylase activation and the myothermally determined energy turnover per twitch, and these showed, identically, that the enzyme activation is proportional to the energy per twitch.     

Stiffness and force in activated frog skeletal muscle fibers.

Single fibers, isolated intact from frog skeletal muscles, were held firmly very near to each end by stiff metal clasps fastened to the tendons. The fibers were then placed horizontally between two steel hooks inserted in eyelets of the tendon clasps. One hook was attached to a capacitance gauge force transducer (resonance frequency up to approximately 50 kHz) and the other was attached to a moving-coil length changer. This allowed us to impose small, rapid releases (complete in less than 0.15 ms) and high frequency oscillations (up to 13 kHz) to one end of a resting or contracting fiber and measure the consequences at the other end with fast time resolution at 4 to 6 degrees C. The stiffness of short fibers (1.8-2.6 mm) was determined directly from the ratio of force to length variations produced by the length changer. The resonance frequency of short fibers was so high (approximately 40 kHz) that intrinsic oscillations were not detectably excited. The stiffness of long fibers, on the other hand, was calculated from measurement of the mechanical resonance frequency of a fiber. Using both short and long fibers, we measured the sinusoids of force at one end of a contracting fiber that were produced by relatively small sinusoidal length changes at the other end. The amplitudes of the sinusoidal length changes were small compared with the size of step changes that produce nonlinear force-extension relations. The sinusoids of force from long fibers changed amplitude and shifted phase with changes in oscillation frequency in a manner expected of a transmission line composed of mass, compliance, and viscosity, similar to that modelled by (Ford, L. E., A. F. Huxley, and R. M. Simmons, 1981, J. Physiol. (Lond.), 311:219-249). A rapid release during the plateau of tetanic tension in short fibers caused a fall in force and stiffness, a relative change in stiffness that putatively was much smaller than that of force. Our results are, for the most part, consistent with the cross-bridge model of force generation proposed by Huxley, A. F., and R. M. Simmons (1971, Nature (Lond.), 213:533-538). However, stiffness in short fibers developed markedly faster than force during the tetanus rise. Thus our findings show the presence of one or more noteworthy cross-bridge states at the onset and during the rise of active tension towards a plateau in that attachment apparently is followed by a relatively long delay before force generation occurs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ratliver cells in culture: Effect of storage,long-term culture,and transformation on some enzyme levels

Summary Aryl hydrocarbon hydroxylase (AHH) and tyrosine aminotransferase (TAT) activities were determined in rat liver cell lines after frozen storage, long-term culture, and transformation in vitro. Levels of AHH activity after 17 months in frozen storage were comparable to levels prior to freezing. During long-term culture the AHH levels of the cell lines tended to decrease. Transformed lines had variable levels of AHH activity. Cell lines retained measurable TAT activity following long-term culture and frozen storage. TAT activity of transformed cells was comparable to that of normal lines. Prolonged frozen storage did not induce transformation up to one year.     

Stiffness, force, and sarcomere shortening during a twitch in frog semitendinosus muscle bundles

The time course of force and stiffness during a twitch was determined at 6 and 26 degrees C in frog semitendinosus muscle bundles using the transmission time technique of Schoenberg, M., J.B. Wells, and R.J. Podolsky, 1974, J. Gen. Physiol. 64:623-642. Sarcomere shortening due to series compliance was also measured using a laser light diffraction technique. Following stimulation, stiffness developed more rapidly than force, but had a slower time course than published Ca2+ transients determined from light signals using Ca2+ sensitive dyes (Baylor, S.M., W.K. Chandler, and M.W. Marshall, 1982, J. Physiol. (Lond.). 331:139-177). Stiffness (S) did not reach its tetanic value during a twitch at 6 or 26 degrees C, although at 6 degrees C, it approached close to this value with S-twitch/S-tetanus = 0.82 +/- 0.07 (+/- SEM). During relaxation, force fell more rapidly than stiffness both for a twitch and also a tetanus. Also in this paper, several of the assumptions inherent in using the transmission time technique for the measurement of stiffness are considered in detail.     

Kinetics of contractile activation in voltage clamped frog skeletal muscle fibers.

Excitation-contraction coupling events leading to the onset of contraction were studied in single skeletal frog muscle fibers. This entailed the simultaneous measurement of the changes in intracellular calcium concentration using antipyrylazo III and fura-2, isometric force, and clamp voltage in a modified single vaseline gap chamber for the first time. The calcium transients were incorporated into an analysis of calcium binding to regulatory sites of troponin C (TnC) that permitted both a linear and a cooperative interaction. The analysis assumed that the onset of mechanical activation corresponds with a particular TnC saturation with calcium setting constraints for the calcium binding parameters of the regulatory sites. Using a simple model that successfully reproduced both the time course and the relative amplitudes of the measured isometric force transients over a wide membrane potential range, k(off) of TnC was calculated to be 78 s(-1) for the cooperative model at 10 degrees C. Together with the above constraints this gave a dissociation constant of 8.8 +/- 2.5 microM and a relative TnC saturation at the threshold (Sth) that would cause just detectable movement of 0.17 +/- 0.03 (n = 13; mean +/- SE). The predictions were found to be independent of the history of calcium binding to the regulatory sites. The observed delay between reaching Sth and the onset of fiber movement (8.7 +/- 1.0 ms; mean +/- SE, n = 37; from seven fibers) was independent of the membrane potential giving an upper estimate for the delay in myofilament activation. We thus emerge with quantitative values for the calcium binding to the regulatory sites on TnC under maintained structural conditions close to those in vivo.     

Active force inhibition and stretch-induced force enhancement in frog muscle treated with BDM.

There is evidence that the stretch-induced residual force enhancement observed in skeletal muscles is associated with 1) cross-bridge dynamics and 2) an increase in passive force. The purpose of this study was to characterize the total and passive force enhancement and to evaluate whether these phenomena may be associated with a slow detachment of cross bridges. Single fibers from frog lumbrical muscles were placed at a length 20% longer than the plateau of the force-length relationship, and active and passive stretches (amplitudes of 5 and 10% of fiber length and at a speed of 40% fiber length/s) were performed. Experiments were conducted in Ringer solution and with the addition of 2, 5, and 10 mM of 2,3-butanedione monoxime (BDM), a cross-bridge inhibitor. The steady-state active and passive isometric forces after stretch of an activated fiber were higher than the corresponding forces measured after isometric contractions or passive stretches. BDM decreased the absolute isometric force and increased the total force enhancement in all conditions investigated. These results suggest that total force enhancement is directly associated with cross-bridge kinetics. Addition of 2 mM BDM did not change the passive force enhancement after 5 and 10% stretches. Addition of 5 and 10 mM did not change (5% stretches) or increased (10% stretches) the passive force enhancement. Increasing stretch amplitudes and increasing concentrations of BDM caused relaxation after stretch to be slower, and because passive force enhancement is increased at the greatest stretch amplitudes and the highest BDM concentrations, it appears that passive force enhancement may be related to slow-detaching cross bridges.     

Differential effects of temperature on contractile behavior in isolated frog skeletal muscle.

1. The thermal dependence of contractile behavior at different stimulation frequencies was investigated in isolated frog sartorius muscles. 2. Increasing incubation temperature (10-30 degrees C) produced decreases in Pt (43.7%) and P15 (70.3%), and an increase in Po (26.0%). 3. Thermal ratios (R10) calculated for Pt, P15 and Po indicated high thermal dependence at lower temperatures (10-20 degrees C; 0.60, 0.44 and 1.38, respectively) but relative thermal independence at higher temperatures (20-30 degrees C; 0.95, 0.75 and 0.95, respectively). 4. Contractile ratios (Pt/Po and P15/Po) decreased with increased temperature (10-30 degrees C; 56.3% and 76.0%, respectively). 5. Thermal ratios (R20) calculated for peak tension at different stimulation frequencies demonstrated high thermal dependence at the lower frequencies (10-30 pps, 0.46-0.48) and decreasing dependence at higher frequencies (40-50 pps, 0.69-0.82).     

The temperature dependence of ionic influences on contractile function in frog twitch muscle

Alterations in the ionic composition of the medium produce striking changes in the potential-dependent contractile responses of skeletal muscles. This study was undertaken to examine the temperature dependence of some of these effects. The suppression of maximal K contractures of frog toe muscles in media lacking divalent cations was largely overcome by a sufficient increase in temperature. The restoration of K contractures by perchlorate in the absence of divalent cations was prevented by a sufficient decrease in temperature. The effect of perchlorate was to shift the temperature dependence of these contractures toward lower temperatures. The reduction in the amplitude of maximal K contractures in the absence of divalent cations was less marked after pretreatment with a reagent (trinitrobenzenesulfonate) that selectively modifies free amino groups, although the temperature dependence of these contractures was unchanged. The reduction in the amplitude of K contractures in the presence of an organic anion (hexanoate) was partially antagonized both by an increase in temperature or by a decrease in temperature, effects that resemble those observed in solutions in which the divalent cation concentration was reduced. In chloride solutions, the relation between [K]0 and K contractures was shifted toward lower [K]0 by an increase in temperature, whereas in perchlorate solutions increased temperature produced a shift in the opposite direction. The shift in this relation toward lower [K]0 at reduced temperature, and the accelerated time course of K contractures with an increase in temperature were similar in perchlorate and in chloride solutions. Thermodynamic analysis by Arrhenius plots indicated that the influence of divalent cations and perchlorate anions on K contractures may be the result of their effects on hydrational factors.     

Rat liver cells in culture: effect of storage, long-term culture, and transformation on some enzyme levels.

Aryl hydrocarbon hydroxylase (AHH) and tyrosine aminotransferase (TAT) activities were determined in rat liver cell lines after frozen storage, long-term culture, and transformation in vitro. Levels of AHH activity after 17 months in frozen storage were comparable to levels prior to freezing. During long-term culture the AHH levels of the cell lines tended to decrease. Transformed lines had variable levels of AHH activity. Cell lines retained measurable TAT activity following long-term culture and frozen storage. TAT activity of transformed cells was comparable to that of normal lines. Prolonged frozen storage did not induce transformation up to one year.     

Cross-linking within the thick filaments of muscle and its effect on contractile force

We have examined the effect of cross-linking on cross-bridge movement and isometric force in glycerinated psoas fibers. Two different methods, high-porosity gel electrophoresis and a fractionation technique, were used to follow the cross-linking of myosin heads (subfragment 1) and rod segments to the thick filament backbone. Contrary to earlier reports [Sutoh, K., & Harrington, W. F. (1977) Biochemistry 16, 2441-2449; Sutoh, K., Chiao, Y. C., & Harrington, W. F. (1978) Biochemistry 17, 1234-1239; Chiao, Y. C., & Harrington, W. F. (1979) Biochemistry 18, 959-963], we find that the heads of the myosin molecules are not cross-linked to the thick filament surface by dimethyl suberimidate. The time dependence of cross-linking rod segments within the core was monitored by a disulfide oxidation procedure to distinguish between intermolecular and intramolecular cross-linking. Comparison of the extent of the cross-linking reaction within myofibrils and the isometric force developed within fibers at various stages of cross-linking shows that isometric force is abolished in parallel with the formation of high molecular weight (cross-linked) rod species (greater than or equal to Mr 1000K). The myofibrillar ATPase remains virtually unaffected by the cross-linking reaction.     

The N-terminal region of twitchin binds thick and thin contractile filaments: redundant mechanisms of catch force maintenance

Catch force maintenance in invertebrate smooth muscles is probably mediated by a force-bearing tether other than myosin cross-bridges between thick and thin filaments. The phosphorylation state of the mini-titin twitchin controls catch. The C-terminal phosphorylation site (D2) of twitchin with its flanking Ig domains forms a phosphorylation-sensitive complex with actin and myosin, suggesting that twitchin is the tether (Funabara, D., Osawa, R., Ueda, M., Kanoh, S., Hartshorne, D. J., and Watabe, S. (2009) J. Biol. Chem. 284, 18015-18020). Here we show that a region near the N terminus of twitchin also interacts with thick and thin filaments from Mytilus anterior byssus retractor muscles. Both a recombinant protein, including the D1 and DX phosphorylation sites with flanking 7th and 8th Ig domains, and a protein containing just the linker region bind to thin filaments with about a 1:1 mol ratio to actin and K(d) values of 1 and 15 μM, respectively. Both proteins show a decrease in binding when phosphorylated. The unphosphorylated proteins increase force in partially activated permeabilized muscles, suggesting that they are sufficient to tether thick and thin filaments. There are two sites of thin filament interaction in this region because both a 52-residue peptide surrounding the DX site and a 47-residue peptide surrounding the D1 site show phosphorylation-dependent binding to thin filaments. The peptides relax catch force, confirming the region's central role in the mechanism of catch. The multiple sites of thin filament interaction in the N terminus of twitchin in addition to those in the C terminus provide an especially secure and redundant mechanical link between thick and thin filaments in catch.     

Stretch-activated force shedding, force recovery, and cytoskeletal remodeling in contractile fibroblasts

The stress fiber network within contractile fibroblasts structurally reinforces and provides tension, or "tone", to tissues such as those found in healing wounds. Stress fibers have previously been observed to polymerize in response to mechanical forces. We observed that, when stretched sufficiently, contractile fibroblasts diminished the mechanical tractions they exert on their environment through depolymerization of actin filaments then restored tissue tension and rebuilt actin stress fibers through staged Ca(++)-dependent processes. These staged Ca(++)-modulated contractions consisted of a rapid phase that ended less than a minute after stretching, a plateau of inactivity, and a final gradual phase that required several minutes to complete. Active contractile forces during recovery scaled with the degree of rebuilding of the actin cytoskeleton. This complementary action demonstrates a programmed regulatory mechanism that protects cells from excessive stretch through choreographed active mechanical and biochemical healing responses.     

Residual force enhancement after stretch of contracting frog single muscle fibers

Single fibers from the tibialis anterior muscle of Rana temporaria at 0.8-3.8 degrees C were subjected to long tetani lasting up to 8 s. Stretch of the fiber early in the tetanus caused an enhancement of force above the isometric control level which decayed only slowly and stayed higher throughout the contraction. This residual enhancement was uninfluenced by velocity of stretch and occurred only on the descending limb of the length-tension curve. The absolute magnitude of the effect increased with sarcomere length to a maximum at approximately 2.9 micrometers and then declined. The phenomenon was further characterized by its dependence on the amplitude of stretch. The final force level reached after stretch was usually higher than the isometric force level corresponding to the starting length of the stretch. The possibility that the phenomenon was caused by nonuniformity of sarcomere length along the fiber was examined by (a) laser diffraction studies that showed sarcomere stretch at all locations and (b) studies of 9-10 segments of approximately 0.6-0.7 mm along the entire fiber, which all elongated during stretch. Length-clamped segments showed residual force enhancement after stretch when compared with the tetanus produced by the same segment held at the short length as well as at the long length. It is concluded that residual force enhancement after stretch is a property shown by all individual segments along the fiber.