Force-velocity relation in chemically skinned rat portal vein

Rat portal veins were chemically skinned using Triton X-100 and mounted for isometric and quick release experiments at 20°C. The skinned preparations were activated by Ca²⁺ (EGTA-buffered) in solutions containing 2 mM free-Mg²⁺ and 1 M calmodulin. Half maximal isometric force was obtained at pCa=6.2. Maximal force of the skinned preparations, at pCa=4.5, was 8.2±0.8 mN/mm² (n=6). Force-velocity relations were determined at varied Ca²⁺-concentrations. Maximal shortening velocity (V _max) was 0.10±0.01 lengths/s at pCa=4.5. At decreasing Ca²⁺-levelsV _max decreased (at pCa=6.25,V _max=0.05 l/s). At pCa =9 an increased level of free-Mg²⁺ (15mM) induces a slow and submaximal increase in tension. Force velocity relations of Mg²⁺-induced contractures were not different from those of Ca²⁺-contractures of similar magnitude (pCa=6.3). The results indicate that the degree of activation of the contractile system, as regulated by Ca²⁺ and Mg²⁺, influences the kinetic properties of the actomyosin interaction as well as the force development.     

Myosin light chain phosphorylation and the cross-bridge cycle at low substrate concentration in chemically skinned guinea pigTaenia coli

Force-velocity relations, rate of ATP turnover (J_ATP), and phosphorylation of the 20,000 D myosin light chains (LC₂₀) were measured in chemically skinned guinea pigTaenia coli. Relative LC₂₀ phosphorylation at 3.2 mM MgATP was 17% in relaxed tissues at pCa 9, and increased with force at increasing [Ca²⁺] to a maximum of 67% at pCa 4.5. Force at pCa 4.5 was dependent on the MgATP concentration with a half-maximal response at about 0.1 mM. At 0.1 mM MgATP LC₂₀ phosphorylation at pCa 4.5 was 38%. Both J_ATP and the maximal shortening velocity (V _max) were reduced in 0.1 mM MgATP, to 32% and 43%, respectively, of their values at 3.2 mM MgATP. Low-MgATP thus inhibits both LC₂₀ phosphorylation and the extent and rate of cross-bridge interaction. High levels of LC₂₀ phosphorylation, independent of Ca²⁺ and MgATP concentrations, were obtained by treatment with ATP--S. Maximal force at 3.2 mM MgATP after LC₂₀ thiophosphorylation was unchanged, whereas halfmaximal force occurred at 0.065 mM MgATP after thiophosphrylation, compared to 0.13 mM after activation by Ca²⁺. The contraction in thiophosphorylated preparations at low-MgATP (0.1 mM) was associated with submaximalV _max (60%) and J_ATP (27%). The results show that LC₂₀ phosphorylation is correlated with the degree of force development in the Ca²⁺ activated contraction, both when Ca²⁺ and MgATP concentrations are varied. The reduced force and rate of crossbridge turnover in lowMgATP are however primarily mediated by an influence of MgATP on the cross-bridge cycle, which is separate from the effect on LC₂₀ phosphorylation.     

Temporal relationship between force,ATPase activity,and myosin phosphorylation during a contraction/relaxation cycle in a skinned smooth muscle

The temporal relationship between myosin phosphorylation, contractile force and ATPase activity was studied in skinned preparations from the guinea-pig Taenia coli. When free Calcium concentration ([Ca²⁺]) was increased from pCa (–log[Ca²⁺]) 9 to pCa 4.5 at low calmodulin concentration (0.05 M), ATPase activity and myosin light-chain phosphorylation rose quickly, while the increase in force and stiffness was delayed. The time-course of tension increase was faster at higher calmodulin concentrations (5 M), although the maximal level of phosphorylation was unchanged. Lowering the calcium concentration from pCa 4.5 to pCa 9 at the plateau of contraction caused a rapid decrease in ATPase activity and in myosin phosphorylation, while force and stiffness decayed more slowly. The force decay could be accelerated by inorganic phosphate. These results suggest that, during contraction, force may be produced actively by phosphorylated and ATP-splitting cross-bridges, but may be maintained by dephosphorylated cross-bridges which cycle slowly. However, force could also be modulated by calmodulin and inorganic phosphate in a manner not involving an alteration in the extent of myosin phosphorylation.     

The effect of calcium on the maximum velocity of shortening in skinned skeletal muscle fibres of the rabbit

Summary The relationship between maximum shortening velocity (V _max) and free calcium concentration has been studied in skinned single fibres from rabbit psoas and soleus muscles. At both 10 and 15° C,V _max measured in the psoas fibres was found to decrease by 40% when the pCa (-log[Ca²⁺]) was increased from 5.49 (maximally activating) to 6.21. Further decreases inV _max were observed when the pCa was increased to 6.32.V _max measured in soleus fibres at 15° C also decreased when the Ca²⁺ concentration was lowered, though the magnitude of this effect was slightly less than in the psoas fibres. Thus, a distinct effect of Ca²⁺ uponV _max has been shown to occur in mammalian skeletal muscle. The occurrence of this effect in both fast and slow muscle types may indicate that the underlying mechanism in the two cases is similar.     

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.     

Effects of 2,3-butanedione monoxime on activation of contraction and crossbridge kinetics in intact and chemically skinned smooth muscle fibres from guinea pig taenia coli

Summary The effects of 2,3-butanedione monoxime (BDM) were studied in smooth muscle fibres from guinea pig taenia coli. In intact muscle, active force during contractions induced by high-K⁺ was inhibited by about 10% in 1 mM BDM and by approximately 70% in 10 mM BDM. Intracellular [Ca²⁺] during contraction, measured with the fura-2 technique, was reduced in the presence of BDM. The reduction in force and [Ca²⁺] in the presence of 1 and 10 mM BDM could be reproduced by reduction in extracellular Ca²⁺, suggesting that BDM influences the Ca²⁺ entry or release. In skinned muscle preparations, BDM decreased the Ca²⁺ sensitivity of active force. This change could be explained by a decreased level of myosin light chain phosphorylation. In fibres maximally activated by thiophosphorylation, the effect of BDM on force occurred at higher concentrations; 10 mM gave no reduction of force and 60 mM 15% reduction. The maximal shortening velocity (V _max) and force were unaffected by 30 mM BDM in thiophosphorylated muscle and decreased almost in parallel in Ca²⁺-activated contractions. The present results suggest that BDM inhibits myosin light chain phosphorylation, directly decreases force generation at the crossbridge level and inhibits the Ca²⁺ translocation in smooth muscle. The effect on force in skinned fibres is observed at higher BDM concentrations than those reported to be required for inhibition of force in striated muscle. The inhibition of force in intact smooth muscle could be explained by an influence on Ca²⁺ translocation.     

Mechanical characteristics of chemically skinned guinea-pig taenia coli

Strips of intact and chemically skinned (Triton X-100) taenia coli were mounted for isometric and quick-release experiments at 23°C. Active force increased in repeated high-K⁺ induced contractures in the intact muscle. Stable maximal force was 313±24 mN/mm² (n=6). The skinned preparations activated by Ca²⁺, at 2 mM Mg²⁺, 3.2 mM MgATP and ionic strength 0.085 M, gave half maximal force atpCa=5.62±0.4 and a maximal force (63±8 mN/mm²) atpCa=4.5 (20–25 of the control K⁺-responses prior to skinning but about 60% of the first K⁺-response). Force-velocity relations were obtained from intact muscles and from the same muscles chemically skinned and activated at optimal Ca²⁺. Maximal shortening velocity (V _max) was unaltered in the skinned preparation compared to the intact muscle (0.138±0.011 vs 0.140±0.006 L/s) indicating similar kinetics of actomyosin interaction. In the intact muscle a decrease inV _max was found when the Ca²⁺ concentration was reduced. Calmodulin (1M) increased Ca²⁺ sensitivity (by about 0.6 log units) of the skinned preparation but at optimal Ca²⁺ caused no alteration in isometric force orV _max Apreliminary report of some of the results presented here was given at the Scandinavian Physiology Society Meeting in Århus, November 1981. Arner A, Hellstrand P (1982) Acta Physiol Scand (Abstract) 114: 38 A.     

Calcium-induced inactivation of calcium release from the sarcoplasmic reticulum of skeletal muscle

The ability of myofilament space Ca²⁺ to modulate Ca²⁺ release from the sarcoplasmic reticulum (SR) of skeletal muscle was investigated. Single fibers of the frog Rana pipiens belindieri were manually skinned (sarcolemma removed). Following a standard load and pre-incubation in varying myoplasmic Ca²⁺ concentrations, SR Ca²⁺ release was initiated by caffeine. Ca²⁺ release rates were calculated from the changes in absorbance of a Ca²⁺ sensitive dye, antipyrylazo III. An apparent dissociation constant (K _d) for dye-Ca²⁺ binding of 8000 M² was determined by comparing the buffering action of the dye with that of ethylenebis(oxonitrilo)tetraacetate (EGTA) using the contractile proteins of the skinned fiber as a measure of free Ca²⁺. This value for K _d was used in the calculation of Ca²⁺ release rates. As the myoplasmic space Ca²⁺ was increased from pCa 7.4, Ca²⁺ release rates declined sharply such that at pCa 6.9 the calculated release rate was 72±3% (mean ± SEM) of control (pCa 8.4). Further increases in myoplasmic Ca²⁺ from pCa 6.9 to pCa 6.1 did not result in a further decline in release rate. The effect of a decreased driving force on Ca²⁺ ions was investigated to determine whether it could account for the change in release rates observed. At pCa 6.9, where the greatest degree of inactivation occurred, the measured effects of a change in driving force could account for at most 40% of the observed inactivation. Varying concentrations of Ba²⁺ and Sr²⁺ in the myofilament space had no inactivating effect on the SR Ca²⁺ release rates. The ability of myofilament Ca²⁺ to inhibit SR Ca²⁺ release at concentrations normally encountered during muscle activation suggests a role for released Ca²⁺ as a modulator of the SR Ca²⁺ channel.     

The contribution of the sarcoplasmic reticulum Ca2+-transport ATPase to caffeine-induced Ca2+ transients of murine skinned skeletal muscle fibres

The present study was carried out to investigate the contribution of the Ca²⁺-transport ATPase of the sarcoplasmic reticulum (SR) to caffeine-induced Ca²⁺ release in skinned skeletal muscle fibres. Chemically skinned fibres of balb-C-mouse EDL (extensor digitorum longus) were exposed for 1 min to a free Ca²⁺ concentration of 0.36 μM to load the SR with Ca²⁺. Release of Ca²⁺ from the SR was induced by 30 mM caffeine and recorded as an isometric force transient. For every preparation a pCa/force relationship was constructed, where pCa = −log₁₀ [Ca²⁺]. In a new experimental approach, we used the pCa/force relationship to transform each force transient directly into a Ca²⁺ transient. The calculated Ca²⁺ transients were fitted by a double exponential function: Y ₀ + A ₁⋅exp (−t/t ₁) + A ₂⋅exp(t/t ₂), with A ₁ < 0 < A ₂, t ₁ < t ₂ and Y ₀, A ₁, A ₂ in micromolar. Ca²⁺ transients in the presence of the SR Ca²⁺-ATPase inhibitor cyclopiazonic acid (CPA) were compared to those obtained in the absence of the drug. We found that inhibition of the SR Ca²⁺-ATPase during caffeine-induced Ca²⁺ release causes an increase in the peak Ca²⁺ concentration in comparison to the control transients. Increasing CPA concentrations prolonged the time-to-peak in a dose-dependent manner, following a Hill curve with a half-maximal value of 6.5 ± 3 μM CPA and a Hill slope of 1.1 ± 0.2, saturating at 100 μM. The effects of CPA could be simulated by an extended three-compartment model representing the SR, the myofilament space and the external bathing solution. In terms of this model, the SR Ca²⁺-ATPase influences the Ca²⁺ gradient across the SR membrane in particular during the early stages of the Ca²⁺ transient, whereas the subsequent relaxation is governed by diffusional loss of Ca²⁺ into the bathing solution. Received: 2 February 1996/Accepted: 1 April 1996     

Effects of vanadate, phosphate and 2,3-butanedione monoxime (BDM) on skinned molluscan catch muscle

The effects of orthovanadate (V_i), inorganic phosphate (P_i) and 2,3-butanedione monoxime (BDM) on tension, force transients and the catch state (passive tension maintenance) were investigated in saponin-skinned fibre bundles of the anterior byssus retractor muscle (ABRM) of the bivalve mollusc Mytilus edulis at pH 6.7. During maximal Ca²⁺ activation isometric force was depressed by V_i (0.03–10 mM), P_i (10 mM) and BDM (50 mM). Force transients following quick stretches (0.1–0.3% of fibre length) were accelerated substantially by 1 mM V_i, 10 mM P_i or 50 mM BDM. These compounds also accelerated force responses in experiments in which ATP was released rapidly from caged ATP by flash photolysis at both pCa 4.7 (force rise) and at pCa>8 (force decline). The effects on the catch state were investigated in two types of experiments: (1) Ca²⁺ removal after maximal Ca²⁺ activation and (2) rapid ATP release during high-force rigor at pCa>8. In both cases rapid relaxation was followed by slow relaxation (slower than 2% of initial force per min). This later slow relaxation (catch) was insensitive to V_i (1–10 mM), P_i (10 mM) and BDM (50 mM) but was accelerated by 0.12 mM cAMP. Complete relaxation to almost zero force was attained by changing pH from 6.7 to 7.7 (pCa>8). We conclude that catch depends on cAMP- and pH-sensitive structures linking the myofilaments and not on the force-generating actomyosin cross-bridges that are sensitive to V_i, P_i and BDM.     

Calcium sensitivity during staircase with sequential incompletely fused contractions

Activity dependent potentiation is thought to result from phosphorylation of the regulatory light chains of myosin, increasing Ca²⁺ sensitivity. Yet, Ca²⁺ sensitivity decreases early in a period of intermittent contractions. The purpose of this study was to investigate the early change in Ca²⁺ sensitivity during intermittent submaximal tetanic contractions. Flexor digitorum brevis muscle fibres were dissected from mice after cervical disarticulation. Fibres were superfused with Tyrode solution at 32 °C. Length was set to yield maximal tetanic force. Indo-1 was microinjected into fibres and allowed to dissipate for 30 min. Fluorescence was measured at 405 and 495 nm wavelength and the ratio was used to estimate [Ca²⁺]. A control force-Ca²⁺ relationship was determined with stimulation over a range of frequencies, yielding constants for slope, max force, and half-maximal [Ca²⁺] (pCa^2?+₅₀). Data were collected for sequential contractions at 40 Hz at 2 s intervals. Active force decreased over the first 1–4 contractions then increased. A force-pCa²⁺ curve was fit to each contraction, using the control values for the Hill slope and max force by adjusting pCa²⁺₅₀ until the curve passed through the target contraction. Data are presented for three contractions for each fibre: first, maximum shift to the right, and last contraction. There was a significant shift to the right for pCa²⁺₅₀ (decreased Ca²⁺ sensitivity), usually early in the series of intermittent contractions, then pCa^2?+₅₀ shifted to the left, but remained significantly different from the control value. Although potentiation is associated with increased Ca²⁺ sensitivity, this increase begins only after Ca²⁺ sensitivity has decreased and, in most cases, Ca²⁺ sensitivity does not increase above the control level.

     

Contractile activation properties of ventricular myocardium from hypothyroid,euthyroid and juvenile rats

Contractile activation properties of intact and chemically skinned ventricular myocardium preparations were studied in juvenile (3–4 weeks old), adult euthyroid and adult hypothyroid rats. The rats were made hyperthyroid by treatment with iodine-131 and propylthiouracil. The ventricular muscle of euthyroid rats contains a mixture of isozymes of myosin while the myocardium of juvenile and hypothyroid rats are relatively pure in regard to V₁ and V₃ types of myosin respectively. No significant differences were found in either the maximum Ca²⁺ activated or rigor force developed by chemically skinned preparations in either the juvenile or hypothyroid groups compared with euthyroid adults, suggesting that there is no difference between myocardia with different isozymes of myosin in the intrinsic capacity to generate force. In the hypothyroid (V₃) preparations there was a significant shift in the force/pCa relation to the left compared with the euthyroid adult (mixture of V₁ and V₃ isozymes). The force/pCa relation for the juvenile lay in between that for the hypothyroid and euthyroid adults. The greater apparent Ca²⁺ sensitivity to activation in the hypothyroid group may relate to a slower cross-bridge cycling rate or altered Ca²⁺ kinetics in ventricular myocardium with exclusively V₃ isozyme. In intact papillary muscles differences were found in the dependence efforce on extracellular [Ca²⁺] such that a higher extracellular [Ca²⁺] was required for muscles from hypothyroid animals to attain maximum twitch force than those from juveniles. The force/frequency relations also differed, with the hypothyroid group being better able to sustain force as stimulation frequency increased than the juvenile group. Also, in the hypothyroid group, the contraction following a 3-min period of quiescence was potentiated in relation to the preceding steady-state contractions, whereas in the juvenile group it was not. These results indicate that the thyroid state may influence the pattern of calcium translocation as well as the myosin isozyme type and that both of these factors may influence contractile properties. Furthermore, the pattern of responses seen depends not only on the pattern of isozymes present but may also depend on the age of the animal because there was no simple relationship between the apparent sensitivity of the contractile apparatus for calcium and proportion of isozyme in the myocardium.     

Compared properties of the contractile system of skinned slow and fast rat muscle fibres

Chemically skinned fibres from soleus and plantaris rat muscles were used to compare the contractile properties of slow and fast muscles. The maximal isometric tension appeared larger in plantaris than in soleus fibres. The apparent Ca²⁺ threshold for activation was lower in slow than in fast fibres while Ca²⁺ concentrations required to obtain either the maximal tension or half maximal tension (pCa₅₀) were lower in fast than in slow fibres. This apparent difference in Ca²⁺ sensitivity will be discussed. As could be expected from other studies, a faster force development in plantaris than in soleus fibres occurred. However, one interesting new result showed that in soleus, the kinetics of the tension development estimated by the t _max parameter were slightly dependent on the Ca²⁺ concentration whereas the t ₅₀ parameter changed significantly with the Ca²⁺ concentration. In plantaris, both t _max and t ₅₀ parameters were found to depend strongly on the Ca²⁺ concentration. Finally, the plantaris muscle showed a greater caffeine sensitivity than the soleus muscle. All the results suggested that the Ca-regulatory mechanism in the slow fibres was essentially different from that in the fast fibres.     

Ca2+ sensitizing effects of EMD 53998 after troponin replacement in skinned fibres from porcine atria and ventricles

Skinned fibres from porcine ventricles exhibited a higher Ca²⁺ sensitivity (pCa₅₀, i.e. -log₁₀ Ca²⁺ concentration required for half-maximal activation, for force generation) than atrial fibres. The thiadiazinone derivative EMD 53998 increased Ca²⁺ sensitivity and Ca²⁺ efficacy in both preparations. The drug effect depended on the isoform of troponin (Tn). Using the vanadate method TnI and TnC could be partly extracted and replaced by foreign tropin or by the TnI subunit of added foreign troponins. We investigated the relationship between pCa and force development before and after replacement of TnI with foreign troponin (bovine ventricular troponin, cTn, or rabbit skeletal muscle troponin, sTn) in the presence and absence of EMD 53998. Substitution with bovine cTn increased Ca²⁺ sensitivity to a value characteristic of bovine ventricular skinned fibres (pCa₅₀=5.4) and was further increased by EMD 53998. Substitution with sTn also increased Ca²⁺ sensitivity, but subsequent addition of EMD 53998 caused little further increase in Ca²⁺ sensitivity. Following extraction of TnI with vanadate, skinned fibres contracted in a Ca²⁺-independent manner and failed to relax at a pCa of 8. Relaxation could be induced, however, by bovine ventricular TnI and rabbit skeletal muscle recombinant TnI. This relaxation could be reversed by EMD 53998 (100 M). The Ca²⁺-independent force of contracted fibres could also be depressed by a TnI inhibitory peptide, (cTnI 137–148) and, in addition, this effect was antagonized by EMD 53998. These results suggest that EMD 53998 antagonizes the inhibitory action of TnI, possibly by interfering with the interaction of the TnI inhibitory region with actin.     

Effects of glycine and proline on the calcium activation properties of skinned muscle fibre segments from crayfish and rat

The effects of the polar amino acid glycine (20 mmol l⁻¹) and the non-polar amino acid proline (20 mmol l⁻¹) on Ca²⁺-activated contraction have been examined in four types of striated muscle fibres. Single fibres dissected from the claw muscle of a crustacean (long- and short-sarcomere) and the hindlimb muscles of the rat (slow-twitch from soleus and fast-twitch from extensor digitorum longus) were activated in matched solutions that either contained the amino acid (‘test’) or not (‘control’). The steady-state force produced in these solutions was used to determine the relation between force production and pCa (−log₁₀[Ca²⁺]). The results show that in the concentrations used, glycine and proline had only small effects on the maximum Ca²⁺-activated force, pCa corresponding to 10, 50 and 90% maximum force (pCa₁₀, pCa₅₀, pCa₉₀, respectively) or on the slope of the force-pCa curves in the four different fibre types. The relative lack of effects of glycine and proline on contractile activation would confer a distinct physiological advantage to force production of muscle of Cherax, where the concentrations of glycine and proline vary considerably. Finally, the results show that glycine and proline may be useful to balance control solutions when the effects of other amino acids or zwitterions on contractile activation are examined. This revised version was published online in July 2006 with corrections to the Cover Date.     

Relation between force and calcium ion concentration in different fibre types of the iliofibularis muscle ofXenopus laevis

Calcium activated isometric force was measured in segments of single muscle fibres of the iliofibularis muscle ofXenopus laevis skinned by freeze-drying. A subdivision in five different fibre types was made, based on the location of the fibres inside the muscle, fibre diameter and a quantitative histochemical assay for succinate dehydrogenase activity. The Ca²⁺ sensivity was characterized by fitting a Hill curve to the force levels reached at different Ca²⁺ concentrations. The parametern of this equation indicates the steepness and pK the midpoint of this force-pCa relation. A considerable variability in the Ca²⁺ sensitivity characteristics was found between different fibres. The parametern varied between 1.1 and 4.2 while pK varied between 5.5 and 6.6. The distribution of the data indicates the presence of three groups with different Ca²⁺ sensitivity; a group of fibres with low Ca²⁺ sensitivity but with considerable variation of the steepness of the Ca²⁺ sensitivity curves (type 1 fibres), an intermediate group (type 2,3 and 4 fibres) with also considerable variation in steepness of the Ca²⁺ sensitivity curves, in which the lowest values forn are found in type 3 and 4 fibres and a group with high Ca²⁺ sensitivity and lown containing at least one tonic (type 5) fibre. At sub-saturating Ca²⁺ concentrations occasionally a transient decrease of the rate of force development was found which resembled the force oscillation reported for some mammalian muscle fibres.     

Calcium and strontium activation characteristics of skeletal muscle fibres from the small marsupialSminthopsis macroura

Summary Mechanically skinned skeletal muscle fibres from the soleus and tibialis anterior muscles of the small marsupialSminthopsis macroura were activated by Ca²⁺ and Sr²⁺ so that their isometric force properties could be determined. The properties characterized were the shape, slope and positions of the curves generated by plotting isometric force vs. pCa (–log₁₀[Ca²⁺]) and pSr (–log₁₀[Sr²⁺]), the maximum Ca²⁺-activated and Sr²⁺-activated tension (Ncm^–2) and the frequency of force oscillations of myofibrillar origin during submaximal activations. The effect of caffeine on force activation was also studied. Apart from the fibres which exhibited physiological characteristics similar to those observed previously in mammalian fibres, a large proportion of fibres exhibited characteristics or combinations of characteristics which have not previously been described from healthy adult mammals. The results from 32 soleus fibres showed that only 23 could be categorized as either typical fast-twitch or slow-twitch fibres. The rest possessed unusual physiological characteristics which suggested the co-existence in the same fibre of Ca²⁺-regulatory and contractile properties from different categories of fast-twitch and slow-twitch fibres. We could distinguish two major fast twitch populations of tibialis anterior fibres which occurred in similar proportions. There were significant differences in the maximum tension produced by some of these groups of fibres. The tibialis anterior population fibres produced the highest maximum tension (T _o ^Ca 44.6±4.6 Ncm^–2, n=7) while the soleus combined type fibres produced the lowest maximum tension (T _o ^Ca 18.1±2.1 Ncm^–2, n=8). Our physiological observations of the Ca²⁺-activation and Sr²⁺-activation properties of soleus fibres in this study provide new evidence that there can be combinations of characteristics in single fibres and a continuum of properties between fibre types in normal mammalian skeletal muscles. These animals can therefore be used as a source of fibres with a wide range of properties.     

Lower active force generation and improved fatigue resistance in skeletal muscle from desmin deficient mice

The mechanical effects of the intermediate filament protein desmin was examined in desmin deficient mice (Des−/−) and their wild type control (Des+/+). Active force generation was determined in intact soleus muscles and in skinned single fibres from soleus and psoas. A decreased force generation of skinned muscle fibres from Des−/− mice and a tendency towards decreased active force in intact soleus muscle were detected. Concentrations of the contractile protein actin and myosin were not altered in Des−/− muscles. Ca²⁺-sensitivity of skinned single fibres in Des−/− muscles was unchanged compared to Des+/+. Using a protocol with repeated short tetani an increased fatigue resistance was found in the intact soleus muscles from Des−/− mice. In conclusion, desmin intermediate filaments are required for optimal generation or transmission of active force in skeletal muscle. Although other studies have shown that the desmin intermediate filaments appear to influence Ca²⁺-handling, the Ca²⁺-sensitivity of the contractile filaments is not altered in skeletal muscle of Des−/− mice. Previous studies have reported a switch towards slower myosin isoforms in slow skeletal muscle of Des−/− mice. The increased fatigue resistance show that this change is reflected in the physiological function of the muscle. This revised version was published online in July 2006 with corrections to the Cover Date.     

Endogenous MLC2 phosphorylation and Ca2+-activated force in mechanically skinned skeletal muscle fibres of the rat

A method has been developed for measuring the level of phosphorylation of myosin regulatory light chains (MLC2) by the endogenous myosin light chain kinase in mechanically skinned skeletal muscle fibres. The method was used to characterize the endogeous MLC2 phosphorylation capacity of single fast-twitch fibres from the rat and to investigate the relationship between the endogenous MLC2 phosphorylation and the Ca²⁺-activated force. The results show that (1) about 50% of MLC2 were ³²P-phosphorylated after activation of the skinned fibre preparation by 30 M [Ca²⁺] for longer than 30 s, but that there was variability between fibres; (2) most of the endogenous phosphorylating system diffused out of the skinned fibre preparation after 5 min exposure to an aqueous solution; (3) the MLC2 phosphorylation by the endogenous phosphorylating system followed with a delay of the order of 1–2 s after the sudden rise in [Ca²⁺] from below 10 nM to 30 M; and (4) the sensitivity of the contractile apparatus to Ca²⁺ was markedly increased when the MLC2 were phosphorylated by the endogenous phosphorylating system following a rise in [Ca²⁺]. The K _d for MgATP of the endogenous MLC2 phosphorylating system was estimated to be less than 300 M. These results unequivocally demonstrate that prolonged activation of the fast-twitch muscle fibre leads to increased Ca²⁺ sensitivity of the contractile apparatus and that mechanically skinned fibres can be successfully used to study the regulation of the endogenous MLC2 phosphorylation capacity at single muscle fibre level.     

The apparent rate constant for the dissociation of force generating myosin crossbridges from actin decreases during Ca2+ activation of skinned muscle fibres

Summary The effect of Ca²⁺ activation on the apparent rate constant governing the dissociation of force generating myosin cross-bridges was studied in skinned rabbit adductor magnus fibres (fast-twitch) at 21±1 °C. Simultaneous measurements of Ca²⁺-activated isometric force and ATPase activity were conducted in parallel with simultaneous measurements of DANZ-labelled troponin C (TnC_DANZ) fluorescence and isometric force in fibres whose endogenous troponin C had been partially replaced with TnC_DANZ. The Ca²⁺ activation of isometric force occurred at approximately two times higher Ca²⁺ concentration than did actomyosin ATPase activity at 2.0 mM MgATP. Since increases in both TnC_DANZ fluorescence and ATPase activity occurred over approximately the same Ca²⁺ concentration range at substantially lower concentrations of Ca²⁺ than did force, this data suggests that the TnC_DANZ fluorescence is associated with the Ca²⁺ activation of myosin crossbridge turnover (ATPase) rather than force. According to the model of Huxley (1957) and assuming the hydrolysis of one molecule of ATP per cycle of the crossbridge, the apparent rate constantg _app for the dissociation of force generating myosin crossbridges is proportional to the actomyosin ATPase/isometric force ratio. This measure ofg _app shows approximately a fivefold decrease during Ca²⁺ activation of isometric force. This change ing _app is responsible for separation of the Ca²⁺ sensitivity of the normalized ATPase activity and isometric force curves. If the MgATP concentration is reduced to 0.5 mM, the change ing _app is reduced and consequently the difference in Ca²⁺ sensitivity between normalized steady state ATPase and force is also reduced.The abbreviations used are TnC troponin C - DANZ 5-dimethylaminonapthalene-2-sulphonyl aziridine - TnC_DANZ DANZ-labelled TnC - a the number of half sarcomeres - A the cross-sectional area of the fibre - AP₅A P¹,P⁵-di(adenosine-5)pentaphosphate - EGTA ethyleneglycolbis-(betaaminoethyl ether)-N,N,N,N-tetraacetic acid - F force a muscle fibre develops - f_app apparent rate of formation of force generating myosin crossbridges - F_av the average force per myosin head - Fs steady-state fraction of cycling myosin crossbridges in the force generating state - g _app apparent rate of dissociation of force generating myosin crossbridges - L_1/2s the length of a half sarcomere - LDH lactate dehydrogenase - [M] is the concentration of myosin per fibre volume - NAD nicotinamide adenine dinucleotide - NADH reduced form of NAD - pCa -log₁₀ of the free Ca²⁺ concentration - PEP phosphenol pyruvate - PK pyruvate kinase