Effects of submaximal activation on the determinants of power of chemically skinned rat soleus fibres

Reducing the activating calcium concentration with skinned fibres is known to decrease isometric force and maximal shortening velocity, both of which will reduce the peak power. However, power is also a function of the curvature of the force-velocity relationship, and there is limited information on how changes in activating calcium affect this important property of muscle fibres. Force-velocity relationships of permeabilized single type I fibres from rat soleus muscle were determined using isotonic contractions at 15°C with both maximal (pCa 4.5) and submaximal activation (pCa 5.6). The rate of tension redevelopment (k(tr)), which provides a measure of sum of the apparent rate constants for cross-bridge attachment and detachment (f(app) + g(app)) following a rapid release and restretch, was also measured. Compared with pCa 4.5, specific tension (P(o)) at pCa 5.6 declined by 22 ± 8% (mean ± s.d.) and the maximal velocity of shortening (V(max)) fell by 44 ± 7%, but curvature of the force-velocity relationship (a/P(o)) rose by 47 ± 31%, indicating a less concave relationship. The value of k(tr) declined by 23 ± 7%. The change in a/P(o) reduced the impact of changes in P(o) and V(max) on peak power by approximately 25%. Fitting the data to Huxley's model of cross-bridge action suggests that lower activating calcium decreased both the rate constant for cross-bridge attachment (f) and that for detachment of negatively strained cross-bridges (g(2)). The fact that V(max) (and thus g(2)) changed to a greater extent than k(tr) (f(app) + g(app)) is the reason that reduced activation results in a reduction in curvature of the force-velocity relationship.     

The effect of hypertonicity on force generation in tetanized single fibres from frog skeletal muscle.

1. We compared the tension transient that follows a step change in sarcomere length in normal Ringer solution with that in Ringer solution made hypertonic by the addition of 98 mM sucrose. Steps were applied on tetanized single muscle fibres during either the isometric plateau or the steady force response to lengthening at low speed. Sarcomere length was controlled on selected fibre segments by a striation follower. Analysis is limited to phase 1 (the tension change simultaneous with the length step, mainly due to cross-bridge elasticity) and phase 2 (the quick phase of tension recovery, a manifestation of the cross-bridge elementary force-generating process). 2. At the isometric tetanus plateau the steady force is reduced by 19% in hypertonic solution, and the stiffness is slightly increased. During slow lengthening both steady force and stiffness are similar in normal solution and in hypertonic solution. In hypertonic solution the tension-to-stiffness ratio, a measure of the mean cross-bridge extension before the step, is markedly reduced in isometric conditions (-23%), but not during lengthening (-2%). 3. The plots of instantaneous tension versus the length change during the step show that in hypertonic medium the elasticity of the fibre is almost undamped. Thus the increase in stiffness cannot be attributed to an increase in viscosity. 4. In isometric conditions (T2-T1)/(Ti-T1), the proportion of the initial tension drop recovered at the end of phase 2, is not affected by hypertonicity for releases of moderate and large size (> 2 nm) and is reduced for small releases (< 2 nm) and for stretches. The abscissa intercept of the relation (T2-T1)/(Ti-T1) versus step amplitude is the same in both media. During lengthening, for releases of small and moderate size, (T2-T1)/(Ti-T1) is 20% lower in hypertonic solution. For large releases the slope of the relation is lower so that the abscissa intercept is not changed. 5. The speed of quick tension recovery following a step length change imposed in isometric conditions is slightly depressed in hypertonic solution. The relation between speed of recovery and step amplitude maintains its shape and is shifted downwards. During lengthening, the speed of quick tension recovery in hypertonic solution is less dependent on step amplitude than in normal solution, as if a more linear viscoelasticity is responsible for a large fraction of residual recovery.(ABSTRACT TRUNCATED AT 400 WORDS)     

Myofibrillar ATPase activity and mechanical performance of skinned fibres from rabbit psoas muscle.

1. The relationship between energy turnover and mechanical performance was investigated in chemically skinned single fibres from rabbit psoas muscle at 15 degrees C, pH = 7.1, with MgATP, 5 mM; free Mg2+, 1 mM; ionic strength, 200 mM and sarcomere length, 2.4 microns by measuring force production and myofibrillar ATP turnover during isometric contractions as well as during repetitive changes in length. ATP hydrolysis was stoichiometrically coupled to the breakdown of NADH, which was measured photometrically via the absorption of near UV light at 340 nm. 2. Force and ATPase activity were measured during square-wave length changes of different amplitudes (1-10% of the fibre length, Lo) and different frequencies (2.5-167 Hz). The average force during the length changes was less than the isometric value and decreased with increasing amplitude and frequency. At full activation (pCa 4.5), the isometric ATP turnover rate (+/- S.E.M.) was 2.30 +/- 0.05 s-1 per myosin head. ATP turnover increased monotonically with increasing amplitude as well as with increasing frequency until saturation was reached. The greatest increase observed was 2.4 times the isometric value. 3. Force and ATPase activity were also determined for ramp shortenings followed by fast restretches. The average force decreased with increasing shortening velocity in a hyperbolic fashion. The ATP turnover increased with ramp velocity up to 0.5 L0 s-1 and stayed almost constant (at 2.2 times the isometric value) for larger velocities. 4. Isometric force and ATPase activity both decreased as the calcium concentration was decreased. They did not vary in proportion at low Ca2+ concentrations, but this could largely be accounted for by the presence of a residual, Ca(2+)-dependent, membrane-bound ATPase. At high calcium concentrations ATPase activity during square-wave length changes was higher than the isometric value, but at low calcium concentrations (pCa > 6.1), the ATPase activity during the length changes decreased below the isometric value and reached a minimum of 40% of the isometric level. 5. ATPase activity and average force obtained during changes in length show a high, movement protocol-independent correlation. During the length changes the rate of ATP turnover divided by the average force level (tension cost) was larger than the isometric tension cost. The largest value found, for 10% length changes at 23 Hz, was 17 times the tension cost under isometric conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ca-induced Ca release from the sarcoplasmic reticulum of isolated myofibrillar bundles of barnacle muscle fibres

Isometric tension and aequorin light were recorded from isolated myofibrillar bundles (diameter 0.2 mm) of barnacle muscle fibres to examine Ca release from the sareoplasmic reticulum (SR). Transfer of a bundle from a pCa 6.7 solution to a pCa 5.8 solution, both buffered with 0.1mM EGTA, resulted in a phasic increase in myofibrillar free Ca²⁺ which was superimposed on a slow rise to a steady level and a fast rise in tension. The peak of the free Ca²⁺ response was higher than the free Ca²⁺ in the bulk solution. Treatment of the bundle with the detergent Brij to destroy the SR membranes abolished the phasic rise in Ca²⁺ and considerably reduced the amplitude of contraction. A second challenge of a bundle to the pCa 5.8 solution without prior reloading of the SR Ca store gave a much reduced phasic component. When a pCa 5.8 solution with 1.0 mM EGTA buffering was used, the phasic rise in myofibrillar free Ca²⁺ could not be detected and the rise in tension was four times slower than with 0.1 mM EGTA. The results are consistent with the operation of a Ca-induced Ca release mechanism in the SR membrane of this crustacean muscle.     

Stretch-induced increase in the Ca2+ sensitivity of myofibrillar ATPase activity in skinned fibres from pig ventricles

The increase in force development in the heart with increase in end-diastolic pressure (Frank-Starling mechanism) has been ascribed to an increase in contractile responsiveness of the myofibrils to calcium. We now show that this calcium sensitization is also associated with an increase in calcium responsiveness of the myofibrillar ATPase. Thus, at submaximal Ca activation (pCa 6.0), the ATPase activity of skinned fibres from pig right ventricles is increased from 57.9±4.4% to 70.6±4.4% of the maximal Ca²⁺ activation of ATPase by stretching (by 15%l _o). At maximal Ca²⁺ activation, ATPase was barely altered by stretching. The relationship between ATPase activity of skinned trabecula of pig right ventricle and ATPase-Ca²⁺ concentrations is shifted (by 0.1 pCa unit) to higher pCa values after a stretch-induced increase of the sarcomere length from 2.1 μm to 2.4 μm. The relationship between force and pCa was affected in a similar way by extension. This increased calcium sensitivity is, however, not associated with an alteration in the relationship between ATPase activity and force development (tension cost). In accordance with Brenner's hypothesis, we propose therefore that stretch activation of ATPase is associated with an increase in the apparent rate constant of crossbridge attachment rather than with a decrease in the apparent rate constant of crossbridge detachment.     

Calcium sensitivity and myofibrillar protein isoforms of rat skinned skeletal muscle fibres

We investigated the calcium sensitivity for tension generation of different fibre types and the possible correlation between calcium sensitivity and the presence of distinct regulatory protein and myosin light chain (MLC) isoforms in rat skinned skeletal muscle fibres. Fibre types 1, 2A and 2B were identified by electrophoretic analysis of myosin heavy chain (MHC) isoforms. Fibres showing more than one MHC isoform were discarded. Type 1 fibres from the soleus showed a higher pCa (–log₁₀ [Ca], where [ ] denotes concentration) threshold and a lower slope of pCa/tension curve than type 2 extensor digitorum longus (EDL) fibres; between type 2 fibres, type 2B showed the higher slope of pCa/tension curve. Type 1 fibres from different muscles showed similar calcium sensitivities when containing only the slow set of regulatory proteins and MLC; when both slow and fast isoforms were present, calcium sensitivity shifted toward fast type fibre values. Type 2A fibres from different muscles showed a similar calcium sensitivity, independently of the set (purely fast or mixed) of regulatory proteins and MLC. It is suggested that when both fast and slow isoforms of regulatory proteins and of MLC are present in a muscle fibre, calcium sensitivity is dictated mainly by the fast isoforms.     

Reduced length-dependent cross-bridge recruitment in skinned fiber preparations of human failing myocardium

A depressed activity of myosin ATPase has been described in human failing myocardium. Since alterations in cross-bridge kinetics may affect both systolic and diastolic cardiac function, the present study simultaneously investigated Ca²⁺-dependent tension and actomyosin ATPase activity (MYO) in triton X-skinned fiber preparations of human non-failing (donor hearts, n=8) and failing (dilated cardiomyopathy, n=11) left ventricular myocardium at increasing sarcomeric length (1.9 and 2.1 μm, α-actinin staining). The MYO/tension ratio was analyzed as a parameter characterizing myofibrillar energetics. At a sarcomere length of 1.9 μm, the Ca²⁺ sensitivity of tension was significantly increased in human failing compared to non-failing myocardium. In human non-failing myocardium, maximal Ca²⁺-activated tension [1.9 μm vs. 2.1 μm, 23.7 (1.9) vs. 28.3 (1.9) mN/mm²] and the Ca²⁺ sensitivity of tension [EC₅₀Ca²⁺ (pCa): 5.67 (0.06) vs. 7.07 (0.11)] were increased by increasing sarcomere length. This was accompanied by an enhancement in Ca²⁺-dependent MYO [+72 (11) vs. +101 (9) μM ADP/s] as well as an increase in the Ca²⁺-sensitivity of MYO [EC₅₀Ca²⁺ (pCa): 5.84 (0.08) vs. 6.86 (0.08)]. In human failing myocardium, only Ca²⁺ sensitivity of tension (but not of MYO) increased. Tension cost was increased in failing vs. non-failing tissue [1.9 μm: 4.18 (0.06) vs. 3.53 (0.06) (mN·s)/(mm²·μM ADP); 2.1 μm: 4.28 (0.13) vs. 3.52 (0.05) (mN·s)/(mm²·μM ADP)]. We concluded that, in human failing myocardium, the length-dependent force generation may be blunted due to an already increased Ca²⁺ affinity of troponin C as well as an impairment of length-dependent cross-bridge recruitment. Electronic Publication     

Series elastic properties of skinned muscle fibres in contraction and rigor

Summary Isometric tension of skinned fibres from the frog semitendinosus muscle is sigmoidally related to Ca²⁺ concentration betweenpCa 7 and 6. Stiffness measurements showed that the Ca²⁺-activated tension may be due to recruitment of attached cross-bridges. In the absence of ATP (rigor solution) the skinned fibre develops a rigor tension which reaches about 80–110% of the maximum Ca²⁺-activated tension.However, stiffness measurements showed that in rigor many more cross-bridges are attached to actin at any one moment than in contraction. It was concluded that the force per cross-bridge is 37% smaller in rigor than in contraction.     

What does TnCDANZ fluorescence reveal about the thin filament state?

Fluorescence of skinned psoas fibres reconstituted with the troponin C subunit labelled with the fluorescent probe dansylaziridine (TnC_DANZ) increases upon activation with Ca²⁺. This fluorescence enhancement is due to Ca²⁺ binding to the Ca²⁺-specific binding sites of TnC_DANZ and attachment of cross-bridges to the actin filament. We found that approximately 20% of the enhanced fluorescence signal derived from Ca²⁺ binding to TnC_DANZ and 80% from cross-bridge attachment during maximal activation. Furthermore we studied the effects of different cross-bridge states on TnC_DANZ fluorescence. Weakly bound, non-force-generating cross-bridge states (pCa 8, low ionic strength) and rigor cross-bridges revealed similar effects on the TnC_DANZ fluorescence. Strongly attached, force-generating states, however, increased fluorescence to the greatest extent. These results suggests a complex system of reciprocal couplings between TnC and different attached cross-bridge states. Cooling or increase of inorganic phosphate decreased isometric force but hardly decreased fluorescence, suggesting the accumulation of attached cross-bridge states with low tension output.     

Activation dependence of isotonic transient in response to step tension reduction in cardiac muscle segment during barium contracture

Summary To clarify the activation-dependence of dynamic mechanical characteristics of contracting cardiac muscle, we analysed the healthy central segment length (SL) response to step decrease in tension at two different levels of barium contracture (0.2 mM and 0.5 mM Ba²⁺) in rat papillary muscles with a fixed initial SL. The time course of this response is thought to reflect the kinetics of actin-myosin interaction. The muscle was released stepwise from the steady contracture tension (T_c) to new steady tension levels (T_r) of varying magnitudes at 22° C. The SL responses consisted of four phases at T_r/T_c > 0.3. The amplitude of shortening in the second phase, after the initial rapid and minute shortening in the first phase, increased with an increase in amplitude of step tension reduction, and was greater at the higher activation level when compared at an identical amount of T_r/T_c. The fourth phase, after the remarkable lengthening in the third phase, was an extremely slow and minute shortening toward a new steady SL under the new tension. The duration of the second and third phase was quite insensitive to activation level at T_r/T_c > 0.85, but became longer at the higher activation level with larger amounts of tension reduction. The velocity measured from the initial quasi-steady SL shortening in the second phase increased significantly with the increase in activation level. These results are discussed in terms of cross-bridge kinetics underlying the isotonic SL transients at two different activation levels.     

Endothermic force generation in skinned cardiac muscle from rat

Isometric tension responses to rapid temperature jumps (T-jumps) of 2–6°C were examined in skinned muscle fibre bundles isolated from papillary muscles of the rat heart. T-jumps were induced by an infra-red laser pulse (wave length 1.32 m, pulse duration 0.2 ms) obtained from a Nd-YAG laser, which heated the fibres and bathing buffer solution in a 50 l trough; the increased temperature by laser pulse was clamped at the high temperature by a Peltier system (see Ranatunga, 1996). In maximally Ca²⁺-activated (pCa ca. 4.5) fibres, the relationship between tension and temperature was non-linear, the increase of active tension with temperature being more pronounced at lower temperatures (below ca. 20°C). A T-jump at any temperature (range 3–35°C) induced an initial step decrease of tension of variable amplitude (Phase 1), probably due to thermal expansion, and it was followed by a tension transient which resulted in a net rise of tension above the pre-T-jump level. The rate of net rise of tension (Phase 2b or endothermic force generation) was 7–10/s at ca. 12°C and its Q₁₀ was 6.3 (below 25°C). In cases where the step decrease of tension in Phase 1 was prominent, an initial quick tension recovery phase (Phase 2a, 70–100/s at 12°C) that did not contribute to a rise of tension above the pre-T-jump level, was also seen. This phase (Phase 2a) appeared to be similar to the quick tension recovery induced by a small length release and its rate increased with temperature with a Q₁₀ of 1.8. In some cases where Phase 2a was present, a slower tension rise (Phase 3) was seen; its rate (ca. 5/s) was temperature-insensitive. The results show that the rate of endothermic force generation in cardiac fibres is clearly different from that of either fast-twitch or slow-twitch mammalian skeletal muscle fibres; implication of such fibre type-specific differences is discussed in relation to the difficulty in identifying the biochemical step underlying endothermic cross-bridge force generation.     

Cyclic AMP inhibits contractility of detergent treated glycerol extracted cardiac muscle

Glycerinated myocardial fibres treated with a detergent (Lubrol WX) and suspended in ATP salt solution produce half maximum isometric tension at pCa 6.2 (at pH 6.7). After addition of cyclic AMP (1–100 M), the pCa required for half maximum activation is 5.9. c-AMP in concentrations of 1–100 M induces a dose dependent inhibition (up to 40$ at pCa 6), and this effect can be amplified by the phosphodiesterase inhibitor IBMX (3-isobutyl-1-methylxanthine) 10^–4 M. The effect is similar in presence and absence of sodium fluoride 10 mM. Since in detergent treated skinned fibres the cell membrane and the sarcoplasmic reticulum are extracted and since the Ca²⁺ ion concentration was kept constant and buffered, we propose that c-AMP does not act via the cell membrane or the sarcoplasmic reticulum, but via phosphorylation of troponin I. The latter is the only component which becomes phosphorylated in skinned fibres during c-AMP induced relaxation, an effect which is also responsible for the inhibition of actomyosin ATPase at constant Ca²⁺ ion concentration (cf. Ray and England 1976).Dedicated to the sixtieth birthday of Prof. Silvio Weidmann, Berne, Switzerland     

Structure-function relationship of soleus muscle fibres from the rhesus monkey

Functional and structural properties of rhesus monkey skinned fibres were studied in order to examine the relationship between calcium/strontium (Ca/Sr) activation characteristics and protein composition. The fibres were classified according to their Ca/Sr affinity into slow (61%) and fast groups (39%). According to the myosin isoform composition, two additional hybrid types were defined. Thus, four profiles were characterized: two corresponding to slow (S) and fast (F) isoforms and two corresponding to a mixed proportion of slow and fast isoforms. They were called hybrid slow (HS) or hybrid fast (HF) based on the predominant myosin isoform. Tension/pCa parameters and maximal shortening velocities were determined. S fibres showed a higher pCa threshold and affinity as well as shallower slopes of their tension/pCa curve than did F fibres. HS and HF fibres exhibited tension/pCa curves which were positioned close to those of S and F fibres, respectively. No significant difference was observed between S and HS fibres or between F and HF fibres. Maximal shortening velocity values were higher for fibres expressing predominantly fast myosin isoforms. We suggest than when both S and F isoforms of myofibrillar proteins are expressed in a muscle fibre, the functional properties are mainly governed by the predominant isoform.     

Characterization of troponin-C interactions in skinned barnacle muscle: comparison with troponin-C from rabbit striated muscle

Previously it was shown that when troponin-C (TnC) is extracted from barnacle myofibrillar bundles they lose their Ca2+ sensitivity, which can be restored by adding back barnacle TnC (either isoform, BTnC1 or BTnC2). Thus barnacle muscle shows thin filament regulation, as does rabbit psoas skeletal muscle. In this paper we comp are the interactions of barnacle and rabbit fast muscle TnC in their respective muscles. We demonstrate that muscle fibres from the giant barnacle, Balanus nubilus, contain about 186 μm kg−1 muscle tissue of BTnC1 plus BTnC2 compared to about 91 μm kg−1 of TnC in rabbit psoas muscle fibres. Extraction of BTnC is achieved using similar low ionic strength, low divalent ion Ca2+-low Mg2+ conditions which are required for TnC extraction in rabbit psoas skinned muscle fibres; extraction was prevented by 1 mm Mg2+. Full reconstitution of Ca2+-sensitivity was achieved by adding back BTnC (1 + 2, or 2). Reconstitution of barnacle muscle with rabbit fast skeletal TnC (RTnC) was more complex, with partial recovery of Ca2+-sensitivity with reconstitution in the presence of 3 mm Mg2+ and more fully with reconstitution in the presence of activating Ca2+ (pCa 4.0). This suggests that the barnacle TnC-TnI (troponin I) recognition sites may be more complex than in rabbit because the barnacle sites appear to have at least two different conformations or types, in which one recognizes RTnC in the presence of Mg2+ and the other only in the presence of Ca2+ and Mg2+. This is consistent with the presence of several TnI isoforms in barnacle striated myofibrils. RTnC has two C-terminal Ca2+-Mg2+ binding sites that are thought to be involved in the Mg2+-sensitive binding of RTnC in rabbit muscle, yet it has been suggested that this site in barnacle muscle does not bind Mg2+, even though Mg2+ stabilizes BTnC binding in barnacle muscle. Consistent with this stabilizing action of Mg2+, using fluorescent probes IAANS or IAE on isolated BTnC2 we demonstrate that BTnC2 binds both Ca2+ and Mg2+, but the data do not suggest direct competition. Consistent with the C-terminal sites on BTnC being Ca2+-specific, BTnC1+2 could only reconstitute low levels of force (about 1/3) in TnC-extracted rabbit skinned muscle fibers in the presence of pCa 4.0 (not just Mg2+) and only at low ionic strengths (0.09 m). Ca2+-activation of contraction was further examined using fluorescently labelled BTnC2 (labelled with IANBD) incorporated into skinned barnacle myofibrillar bundles. Maximal Ca2+ binding produced structural changes in BTnC which resulted in a 45% decrease in the fluorescence compared to the value at pCa 9.2. The magnitude of the fluoresence decrease paralleled the increase in force with increas ing Ca2+. The Hill fits to the data gave pCa1/2 and n of 5.61 ± 0.02 and 2.06 ± 0.12 for force, and 5.52 ± 0.02 and 1.88 ± 0.10 for fluoresence. Removing MgATP to induce rigor in the fibre decreased BTnC2-NBD fluorescence only about 11%, but the addition of Ca2+ in rigor further decreased the fluorescence to a slightly larger extent than under maximal Ca2+ activating conditions. These fluorescence changes are qualitatively similar to the fluorescence enhancement seen with Ca2+-activation and rigor with RTnCDanz exchanged into rabbit psoas skinned muscle fibres. The data support a similar model for Ca2+-activation of force in barnacle muscle and in rabbit psoas skeletal muscle fibres This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of a preferential myosin loss on Ca2+ activation of force generation in single human skeletal muscle fibres

Preferential loss of the motor protein myosin, as observed in patients with acute quadriplegic myopathy (AQM) or cancer cachexia, causes generalized muscle wasting, muscle weakness and a decrease in muscle fibre force normalized to cross-sectional area. It remains unclear, however, whether this myosin loss influences other important features of muscle fibre function, such as Ca²⁺ activation of the contractile proteins. To address this question, we have studied Ca²⁺ sensitivity of force generation using skinned muscle fibres from four patients with AQM or cancer cachexia and a preferential loss of myosin; and from seven healthy control individuals. Force and apparent rate constant of force redevelopment ( k _tr) were assessed in solutions with varying Ca²⁺ concentrations (pCa), allowing construction of relative force–pCa and k _tr–pCa relationships. Results showed a rightward shift of the relative force–pCa relationship and a leftward shift of the relative k _tr–pCa curve in muscle fibres with a preferential myosin loss. To improve the understanding of the mechanisms underlying these alterations, the relative stiffness–pCa relationship was evaluated. A rightward shift of this curve was observed, suggesting that the changes in the Ca²⁺ activation of force and k _tr were predominantly due to a decrease in the relative number of attached cross-bridges at different pCa values. Thus, a change in Ca²⁺ activation of the contractile apparatus in patients with preferential myosin loss is proposed as an additional factor contributing to the muscle function impairment in these patients.     

Adenosine affects the release of Ca2+ from the sarcoplasmic reticulum via A2A receptors in ferret skinned cardiac fibres

In this study, it was shown that adenosine potentiates caffeine-induced Ca2+ release. It was then proposed that the enhancement of the caffeine-induced Ca2+ release might occur by a direct effect on the ryanodine Ca2+ release channel or on other Ca2+ regulation mechanisms. Furthermore, A2A receptors may be functional on the ferret cardiac sarcoplasmic reticulum. Using chemically skinned fibres, experiments were conducted on ferret cardiac muscle to find out whether adenosine and the A1 and A2A adenosine receptor agonists (CCPA and CGS 21680) and antagonists (DPCPX and ZM 241385) affected caffeine-induced Ca2+ release and the Ca2+ sensitivity of contractile proteins. Changes in the caffeine-induced contracture brought about by adenosine and by adenosine-receptor agonists and antagonists were recorded in saponin-skinned fibres (50 microg ml(-1)). Tension-pCa relationships were then obtained by exposing Triton X-100-skinned fibres (1% v/v) sequentially to solutions of decreasing pCa. Adenosine (1-100 nm) and the specific A2A receptor agonist CGS 21680 (1-50 nm) produced a concentration-dependant potentiation of the caffeine-induced Ca2+ release from saponin-skinned fibres. The data plotted versus adenosine and CGS 21680 concentrations displayed sigmoid relationships (Hill relationship), with potentiation of Ca2+ release by 22.2 +/- 1.6 (n = 6) and 10.9 +/- 0.4% (n = 6), respectively. In addition, the potentiation of caffeine-induced Ca2+ release by adenosine (50 nm; 15.3 +/- 1.0%; n = 6) and by CGS 21680 (50 nm; 11.2 +/- 0.4%; n = 6) was reduced by the specific A2A receptor antagonist ZM 241385 (50 nm) to 8.0 +/- 1.4 (n = 4) and 5.4 +/- 1.2% (n = 4), respectively. The A1 receptor agonist CCPA (1-50 nm) and antagonist DPCPX (50 nm) had no significant effects on caffeine responses. In Triton X-100-skinned fibres, the maximal Ca(2+)-activated tension of the contractile proteins (41.3 +/- 4.1 mN mm(-2); n = 8), the Hill coefficient (nH = 2.2 +/- 0.1; n = 8) and the pCa50 (6.15 +/- 0.05; n = 8) were not significantly modified by adenosine (100 nm) or by CGS 21680 (50 nm).     

Effects of Ca antagonist on the contractile force in glycerinated dog heart muscles

The effect of Ca antagonist on the contractile apparatus was investigated in glycerinated cardiac muscle preparations obtained from canine hearts. Each muscle preparation had three consecutive isometric contractions. The 1st and 3rd contractions were produced with a control contraction solution, and compared with the 2nd contraction which was induced with a contraction solution containing verapamil. The results showed that maximal developed tension (Po) was enhanced significantly by 1.02×10^–2 mM of verapamil, and the augmentation of contractility was dependent on the concentrations of verapamil. Thus, not only Po, but also dT/dt increased tremendously at 1.02 mM of verapamil. Such contractile potentiation by verapamil was also ascertained by another Ca antagonist, Diltiazem hydrochloride. The developed tension was maximum at pCa 4.0, and no developed tension was found at pCa 8.0. The relationship between pCa and tension with verapamil shifted to the left from that without verapamil, showing higher sensitivity to Ca²⁺. From these results, it was strongly indicated that Ca antagonist is a potentiating agent of the contractile force.     

Activation of skinned muscle fibres from the Norway lobster Nephrops norvegicus L. by manganese ions

Effects of Mn2+ and Ca2+ on the mechanical properties of glycerinated myofibrillar bundles originating from slow S1 type muscle fibres of superficial flexor muscles of the lobster Nephrops norvegicus were investigated. Mn2+ (5–20μm) activated the preparations in a dose-dependent manner. The sensitivity of myofibrillar force generation for Mn2+ was around 30 times lower than that for Ca2+. The maximal tension produced under Mn2+ activation was about 75% of that under Ca2+ activation. At higher free Mn2+ concentrations (>2mm), the steady-state force decreased; it was completely abolished at 30mm free Mn2+. These high Mn2+ solutions were accompanied by changes in MgATP and MnATP concentrations, and in the ionic strength. Control experiments have shown that none of these parameters seemed fo account fully for the observed force depression in high Mn2+ solutions. It is likely that direct effects of Mn2+ such as a change of the myofilament surface charges are responsible. The maximal unloaded shortening velocity of the myofibrillar preparations was shown to be similar under maximal Mn2+ and Ca2+ activation. Conversely, the kinetics of stretch-induced delayed force increase were about two to three times faster under Mn2+ activation. These results suggest that certain steps of the cross-bridge cycle depend on the ion species bound to the regulatory proteins. This revised version was published online in July 2006 with corrections to the Cover Date.     

2,3-Butanedione monoxime increases speed of relaxation in single muscle fibres of frog.

The effects of 2,3-butanedione monoxime (BDM) on intracellular Ca2+ transient and cross-bridge function were studied in frog single fibres from the anterior tibialis muscle of Rana temporaria (sarcomere length, 2.2 microm; temperature, 2-4 degrees C). The fluorescent dye fluo-3 was used to monitor the intracellular free calcium concentration ([Ca2+]i) during isometric contractions. BDM (1-5 mM) reduced the amplitude of the Ca2+ transient during twitches, but this effect was too small to explain the marked inhibition of BDM on twitch force. [Ca2+]i reached at the end of 1-s tetanic stimulation was not significantly affected by BDM (1.0 and 1.8 mM) while the maximum tetanic tension was substantially reduced. The rate of relaxation during isometric tetanus was increased by BDM whereas the rate of decay of the Ca2+ transient was reduced in the presence of BDM. The results strongly suggest that BDM, under the experimental conditions used, mainly affects the contractile machinery resulting in altered performance of the cross-bridges. These effects of BDM were evaluated in terms of the cross-bridge model of Huxley (1957) which was fitted to the experimental force-velocity data in the presence and absence of BDM.     

Effects of halothane on functionally skinned rabbit soleus muscle fibers: A correlation between tension transient and45Ca release

The mechanism(s) involved in the halothane-induced increase in skeletal muscle contraction was studied using functionally skinned soleus muscle fibers from rabbits: For the tension study, single functionally skinned fibers were individually mounted on two pairs of forceps, with one end attached to a photodiode tension transducer. Ca²⁺-activated tension development of the contractile proteins, and Ca²⁺ uptake and release from the sarcoplasmic reticulum (SR) using caffeine-induced tension transients were studied. To measure the amount of calcium, skinned fibers at 0.1 g/ml were used and 0.075 Ci⁴⁵Ca/ml was spiked in the solution 3 (pCa 6.5 and 1 mM [EGTA]) which promoted rapid loading of Ca²⁺. Halothane (1–3%) did not change the [Ca²⁺]-tension relationship; 2 and 3% halothane reduced the maximum Ca²⁺-activated tension by 6–7%. Halothane (1–3%) added to the solution 3, reduced⁴⁵Ca uptake by 3, 22 and 23%; however, the subsequent caffeine-induced tension transient and⁴⁵Ca release were increased by 10–40%. During the release phase only halothane increased both caffeine-induced tension transient and⁴⁵Ca release by 20–60%. The effects of halothane on the tension transient and on the⁴⁵Ca release were comparable. There was no dose-response relationship to the effects of halothane on the above parameters. It is concluded that halothane affects the SR by increasing its membrane permeability to Ca²⁺, resulting in an increase in myoplasmic [Ca²⁺] and thus in the twitch tension in skeletal muscle.