Jack A. Rall

Parvalbumin content and Ca2+ and Mg2+ dissociation rates correlated with changes in relaxation rate of frog muscle fibres.

1. Experiments were done to test the hypothesis that parvalbumin (PA) promotes relaxation in frog skeletal muscle. Single fibres and purified PA from Rana temporaria skeletal muscle were used to determine the relationship between PA concentration ( [PA] ), Ca2+ and Mg2+ dissociation rates from PA and changes in rate of relaxation as a function of tetanus duration at 0 degrees C. 2. Total [PA] in fibres from tibialis anterior muscles is 0.76 +/‐ 0.20 mmol PA l‐1 myoplasmic water (mean +/‐ S.D., n = 25) with 65% PA IVa and 35% PA IVb, where PA IVa and PA IVb are PA isoforms. 3. Relaxation rate from an isometric tetanus shows progressively as a function of tetanus duration with an exponential time course and a rate constant of 1.18 +/‐ 0.35 s‐1 (n = 17). Time course of recovery of relaxation rate after a prolonged tetanus is exponential with a rate constant of 0.12 +/‐ 0.02 s‐1 (n = 14). 4. The extent of recovery of relaxation rate after a prolonged tetanus was correlated with total [PA] in fibres (correlation coefficient (r) = 0.80, n = 7; P less than 0.05). 5. Dissociation rate constants for Mg2+ and Ca2+ from purified PA are 0.93 +/‐ 0.02 s‐1 (n = 5) and 0.19 +/‐ 0.01 s‐1 (n = 5), respectively. Dissociation rate constants were not significantly different for PA isoforms IVa and IVb. These rate constants are similar to the rate constants determined for the time courses of slowing and recovery of relaxation rate, respectively. 6. Results suggest that the time courses of slowing and recovery of relaxation rate may be controlled, to a large extent, by Mg2+ and Ca2+ dissociation from PA, respectively. This evidence supports a role for PA in facilitating relaxation during a tetanus in frog skeletal muscle at 0 degrees C.

Effect of temperature on relaxation rate and Ca2+, Mg2+ dissociation rates from parvalbumin of frog muscle fibres.

1. Influence of temperature on relaxation rate as a function of isometric tetanus duration and on Ca2+ and Mg2+ dissociation rates from purified parvalbumin (PA) was examined to test the hypothesis that PA promotes relaxation in frog skeletal muscle. Single fibres and PA IVB from Rana temporaria skeletal muscle were utilized. 2. The magnitude of slowing of relaxation rate with increasing tetanus duration, relative to the final, steady value of relaxation rate, is 3‐fold greater at O than at 10 degrees C. 3. In the 0‐10 degrees C range, the Q10 for relaxation rate increases from 2.3 to 3.7 with increasing tetanus duration. 4. Dissociation of Ca2+ and Mg2+ from PA exhibited: (i) rate constants of 1.03 +/‐ 0.03 s‐1 (mean +/‐ S.D., n = 5) and 3.42 +/‐ 0.14 s‐1 (n = 5) at 20 degrees C and (ii) Q10 values of 2.3 and 1.9 in the 0‐20 degrees C range, respectively. 5. Time courses of slowing of relaxation rate with increasing tetanus duration and recovery of relaxation rate with rest after a prolonged tetanus at 10 degrees C are similar to rates of dissociation of Mg2+ and Ca2+ from PA, respectively, as previously reported at 0 degree C. 6. Both the temperature dependence of the relative magnitude of slowing of relaxation rate and the increased Q10 of relaxation rate with increased tetanus duration can be explained if the Q10 for Ca2+ uptake by the sarcoplasmic reticulum is greater than the Q10 for Ca2+ sequestration by PA during relaxation. When Ca2+ and Mg2+ dissociation rates from PA at various temperatures are compared to other proposed indicators of PA function, it is concluded that PA facilitates relaxation of frog skeletal muscle in the 0‐20 degrees C range.

Determinants of relaxation rate in rabbit skinned skeletal muscle fibres

The influence of Ca2+‐activated force, the rate of dissociation of Ca2+ from troponin C (TnC) and decreased crossbridge detachment rate on the time course of relaxation induced by flash photolysis of diazo‐2 in rabbit skinned psoas fibres was investigated at 15 °C. The rate of relaxation increased as the diazo‐2 chelating capacity (i.e. free [diazo‐2]/free [Ca2+]) increased. At a constant diazo‐2 chelating capacity, the rate of relaxation was independent of the pre‐photolysis Ca2+‐activated force in the range 0.3‐0.8 of maximum isometric force. A TnC mutant that exhibited increased Ca2+ sensitivity caused by a decreased Ca2+ dissociation rate in solution (M82Q TnC) also increased the Ca2+ sensitivity of steady‐state force and decreased the rate of relaxation in fibres by approximately twofold. In contrast, a TnC mutant with decreased Ca2+ sensitivity caused by an increased Ca2+ dissociation rate in solution (NHdel TnC) decreased the Ca2+ sensitivity of steady‐state force but did not accelerate relaxation. Decreasing the rate of crossbridge kinetics by reducing intracellular inorganic phosphate concentration ([Pi]) slowed relaxation by approximately twofold and led to two phases of relaxation, a slow linear phase followed by a fast exponential phase. In fibres, M82Q TnC further slowed relaxation in low [Pi] conditions by approximately twofold, whereas NHdel TnC had no significant effect on relaxation. These results are consistent with the interpretation that the Ca2+‐dissociation rate and crossbridge detachment rate are similar in fast‐twitch skeletal muscle, such that decreasing either rate slows relaxation, but accelerating Ca2+ dissociation has little effect on relaxation.

Determinants of relaxation rate in skinned frog skeletal muscle fibers

The influences of sarcomere uniformity and Ca2+ concentration on the kinetics of relaxation were examined in skinned frog skeletal muscle fibers induced to relax by rapid sequestration of Ca2+ by the photolysis of the Ca2+ chelator, diazo-2, at 10 degreesC. Compared with an intact fiber, diazo-2-induced relaxation exhibited a faster and shorter initial slow phase and a fast phase with a longer tail. Stabilization of the sarcomeres by repeated releases and restretches during force development increased the duration of the slow phase and slowed its kinetics. When force of contraction was decreased by lowering the Ca2+ concentration, the overall kinetics of relaxation was accelerated, with the slow phase being the most sensitive to Ca2+ concentration. Twitchlike contractions were induced by photorelease of Ca2+ from a caged Ca2+ (DM-Nitrophen), with subsequent Ca2+ sequestration by intact sarcoplasmic reticulum or Ca2+ rebinding to caged Ca2+. These twitchlike responses exhibited relaxation kinetics that were about twofold slower than those observed in intact fibers. Results suggest that the slow phase of relaxation is influenced by the degree of sarcomere homogeneity and rate of Ca2+ dissociation from thin filaments. The fast phase of relaxation is in part determined by the level of Ca2+ activation.The influences of sarcomere uniformity and Ca2+ concentration on the kinetics of relaxation were examined in skinned frog skeletal muscle fibers induced to relax by rapid sequestration of Ca2+ by the photolysis of the Ca2+ chelator, diazo-2, at 10°C. Compared with an intact fiber, diazo-2-induced relaxation exhibited a faster and shorter initial slow phase and a fast phase with a longer tail. Stabilization of the sarcomeres by repeated releases and restretches during force development increased the duration of the slow phase and slowed its kinetics. When force of contraction was decreased by lowering the Ca2+concentration, the overall kinetics of relaxation was accelerated, with the slow phase being the most sensitive to Ca2+ concentration. Twitchlike contractions were induced by photorelease of Ca2+ from a caged Ca2+ (DM-Nitrophen), with subsequent Ca2+ sequestration by intact sarcoplasmic reticulum or Ca2+ rebinding to caged Ca2+. These twitchlike responses exhibited relaxation kinetics that were about twofold slower than those observed in intact fibers. Results suggest that the slow phase of relaxation is influenced by the degree of sarcomere homogeneity and rate of Ca2+ dissociation from thin filaments. The fast phase of relaxation is in part determined by the level of Ca2+ activation.

Temperature dependence of the crossbridge cycle during unloaded shortening and maximum isometric tetanus in frog skeletal muscle.

SummaryThe primary objective of this study was to determine if the rate limiting step in the crossbridge cycle was the same during maximum rate of shortening and during maintenance of maximum tension in an isometric contraction. To this end the temperature dependence,Q10, of the crossbridge cycle was estimated during unloaded shortening and maximum isometric tetanus. Isolated semitendinosus muscles from the frog were studied at 0 and 10° C. Crossbridge cycling during unloaded shortening was determined from the velocity of unloaded shortening estimated by the slack step technique. Crossbridge cycling during maintained isometric tetanus was determined from the steady rate of energy liberation during the tetanus after allowance for energy liberation due to Ca2+ cycling. TheQ10 of the velocity of unloaded shortening was 2.5 and that of the steady rate of energy liberation was 4.6. After correction for the temperature dependence of energy liberation associated with Ca2+ cycling (5.7), the estimatedQ10 of the steady rate of energy liberation became 3.9. These estimates of theQw of the crossbridge cycle are significantly different. These results support the conclusion that the rate limiting steps during unloaded shortening and maximum isometric force maintenance occur at different steps in the crossbridge cycle. Further the high Q10 of the energy liberation due to Ca2+ cycling may relate to the high concentration of parvalbumin in frog muscle. A second objective of this study was to document in the same muscle the variation of Q10s of mechanical and energetic properties of contraction. Over this temperature range the Q10s ranged from 1.1 to 5.7.

Myofibrillar determinants of rate of relaxation in skinned skeletal muscle fibers.

The influence of Ca2+ dissociation rate from TnC and decreased cross-bridge detachment rate on the time course of relaxation induced by flash photolysis of diazo-2 in rabbit skinned psoas fibers was investigated at 15 degrees C. A TnC mutant (M82Q TnC) that exhibited increased Ca2+ sensitivity caused by a decreased Ca2+ dissociation rate in solution also increased the Ca2+ sensitivity of force and decreased the rate of relaxation in fibers approximately 2-fold. In contrast, a TnC mutant (NHdel TnC) with decreased Ca2+ sensitivity caused by an increased Ca2+ dissociation rate in solution decreased Ca2+ sensitivity of force but did not accelerate relaxation. Decreasing the rate of cross-bridge kinetics by reducing [Pi] slowed relaxation -2-fold and led to two phases of relaxation, a linear phase followed by an exponential phase. In fibers, M82Q TnC further slowed relaxation in low [Pi] approximately 2-fold whereas NHdel TnC had no significant effect on relaxation. These results are consistent with the interpretation that the Ca2+ dissociation rate and cross-bridge detachment rate are similar in fast twitch skeletal muscle such that decreasing either rate slows relaxation but accelerating Ca2+ dissociation has little effect on relaxation.

Effect of Ca2+ binding properties of troponin C on rate of skeletal muscle force redevelopment.

To investigate effects of altering troponin (Tn)C Ca(2+) binding properties on rate of skeletal muscle contraction, we generated three mutant TnCs with increased or decreased Ca(2+) sensitivities. Ca(2+) binding properties of the regulatory domain of TnC within the Tn complex were characterized by following the fluorescence of an IAANS probe attached onto the endogenous Cys(99) residue of TnC. Compared with IAANS-labeled wild-type Tn complex, V43QTnC, T70DTnC, and I60QTnC exhibited ∼1.9-fold higher, ∼5.0-fold lower, and ∼52-fold lower Ca(2+) sensitivity, respectively, and ∼3.6-fold slower, ∼5.7-fold faster, and ∼21-fold faster Ca(2+) dissociation rate (k(off)), respectively. On the basis of K(d) and k(off), these results suggest that the Ca(2+) association rate to the Tn complex decreased ∼2-fold for I60QTnC and V43QTnC. Constructs were reconstituted into single-skinned rabbit psoas fibers to assess Ca(2+) dependence of force development and rate of force redevelopment (k(tr)) at 15°C, resulting in sensitization of both force and k(tr) to Ca(2+) for V43QTnC, whereas T70DTnC and I60QTnC desensitized force and k(tr) to Ca(2+), I60QTnC causing a greater desensitization. In addition, T70DTnC and I60QTnC depressed both maximal force (F(max)) and maximal k(tr). Although V43QTnC and I60QTnC had drastically different effects on Ca(2+) binding properties of TnC, they both exhibited decreases in cooperativity of force production and elevated k(tr) at force levels <30%F(max) vs. wild-type TnC. However, at matched force levels >30%F(max) k(tr) was similar for all TnC constructs. These results suggest that the TnC mutants primarily affected k(tr) through modulating the level of thin filament activation and not by altering intrinsic cross-bridge cycling properties. To corroborate this, NEM-S1, a non-force-generating cross-bridge analog that activates the thin filament, fully recovered maximal k(tr) for I60QTnC at low Ca(2+) concentration. Thus TnC mutants with altered Ca(2+) binding properties can control the rate of contraction by modulating thin filament activation without directly affecting intrinsic cross-bridge cycling rates.

Absolute values of myothermic measurements on single muscle fibres from frog

SummaryThe heat and force produced in tetanic contraction of single fibres from anterior tibialis muscle of the frogRana temporaria have been observed at measured temperatures close to 1 and 10° C. Heat was measured using a Hill-Downing type thermopile. In control experiments with a resistor of known heat capacity comparable to a single muscle fibre, it was found that Peltier and Joule heating produced identical thermopile outputs. The Peltier method was used to introduce a known amount of heat into the system in each experiment with a muscle fibre. From the response to this heating the heat capacity of each preparation was obtained and used to calculate the absolute amount of heat production from the thermopile output.The heat produced during tetanic contraction (H) could be described by Auberts equation [H=Ha(1−et/τ)+hbt]. In some fibres there was no labile heat (Ha), whereas in others it was clearly present. The stable heat rate (hb) was strongly temperature dependent (Q10 = 4.06). At 0° C the stable heat rate (normalized by dry weight) in the single fibres was significantly greater than that in whole anterior tibialis muscle.

Simultaneous Heat and Tension Measurements from Single Muscle Cells

Simultaneous force and heat measurements were made in single cells from skeletal muscle of the frog during isometric twitches and tetani at 10 and 0 degree C. A Hill- Downing type thermopile of low heat capacity was used. In twitches, peak force development was found to be well correlated with heat production at both temperatures, during posttetanic twitch potentiation (at 10 degrees C) and during posttetanic twitch depression (at 0 degree C). In a twitch at 0 degree C, heat production started less than 14 msec after the stimulus had begun, before force development. As in whole muscle, the heat during a tetanus could be separated into two components: an early component produced at an exponentially decreasing rate, labile heat, and a steady rate, stable maintenance heat rate. Increasing temperature from 0 to 10 degrees C doubled the stable maintenance heat rate. At the higher temperature the time constant of labile heat production was halved and the quantity of labile heat decreased. When two tetani were given at 10 degrees C, a 5 min rest interval was required before the second tetanus produced the same force and heat as the first. At 0 degree C this interval was at least 10 min. With shorter intervals, both heat and force were depressed. At 10 degrees C both were depressed equally but at 0 degree C the effect on heat was greater than on force. At both temperatures labile heat was depressed to a greater extent than the stable maintenance heat rate. Results are interpreted in terms of possible calcium-parvalbumin interaction during a tetanus.

Energetics, Mechanics and Molecular Engineering of Calcium Cycling in Skeletal Muscle

During muscle contraction and relaxation, Ca2+ moves through a cycle. About 20 to 40% of the ATP utilized in a twitch or a tetanus is utilized by the SR Ca2+ pump to sequester Ca2+. Parvalbumin is a soluble Ca2+ binding protein that functions in parallel with the SR Ca2+ pump to promote relaxation in rapidly contracting and relaxing skeletal muscles, especially at low temperatures. The rate of Ca2+ dissociation from troponin C, once thought to be much more rapid than the rate of relaxation, is likely to be similar to the rate of cross-bridge detachment and to the rate of muscle relaxation under some conditions. During the past fifty years, great progress has been made in understanding the Ca2+ cycle during skeletal muscle contraction and relaxation. Nonetheless, there are still mysteries waiting to be unraveled.