Fluorescence changes on contractile activation in TnCDANZ labeled skinned rabbit psoas fibers

The increase in fluorescence of dansylaziridine (DANZ) labeled troponin C (TnC_DANZ) substituted into skinned rabbit psoas fibers was determined as a function of the pCa. The fluorescence data are expressed as the ratio of two wavelength bands, one that sees the fluorescence of TnC_DANZ, and one that sees background fluorescence and scatter. The percent TnC replaced with TnC_DANZ was varied between 10 and 50% and, the fibers were randomly stretched, at the start of each experiment, between 10 and 50%. A large ratio increase accompanies increase in [Ca²⁺]. The pCa/force data are best fit by the Hill equation but the pCa/ratio data are best fit by a model in which Ca²⁺ binds in two phases. The position of the force curve on the pCa axis varies little between fibers, in contrast to that of the ratio or Δ-fluorescence curve. In accord with previous reports the Δ-fluorescence can be left of the force on the pCa axis (type I) or superimpose in part on the force (type II). Not described previously, we find curves in which the second phase of the ratio cross-over the pCa/force curve. This type III relationship is found only in fibers less than 3 weeks postmuscle harvest. We propose that the first, relatively invariant, phase of the biphasic pCa/ratio curve accompanies Ca²⁺ binding to either of the two low affinity sites on TnC_DANZ as it does for TnC in solution. The second, highly cooperative, phase of the ratio curve that accompanies muscle contraction and enhanced Ca²⁺ binding is initiated when sufficient Ca²⁺ is bound to overcome inhibitory systems. Loose coupling between the initial Ca²⁺ binding and the cooperative switch point may account for much of the variation in the shape and position of the pCa/ratio curve. There is evidence that, in the overlap zone, weakly attached myosin cross-bridges enhance cooperation between the regulatory units of the thin filaments. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inositol trisphosphate has no direct effect on the contractile apparatus of skinned cardiac muscles

Alpha-adrenoceptor stimulation produces a positive inotropic effect in heart muscle via mechanisms that are not well understood. The purpose of our study was to test the hypothesis that the increase in inositol 1,4,5 trisphosphate [Ins(1,4,5)P₃] concentration that accompanies alpha stimulation contributes to the inotropic effect by increasing the calcium sensitivity of the contractile proteins, an effect which Ins(1,4,5)P₃ has been shown to have in skeletal muscle. We determined the calcium sensitivity of the contractile apparatus of small, chemically skinned bundles from papillary muscles of rabbit, rat and dog hearts. These preparations were chosen because they exhibit a range of sensitivity to alpha agonists. In addition, we measured the calcium sensitivity of chemically skinned, single fibers from rabbit psoas muscle. All preparations were skinned with Triton X-100, a non-ionic detergent that disrupts the sarcolemmal, sarcoplasmic reticular, and mitochondrial membranes. In all cardiac preparations, we found that 38 M Ins(1,4,5)P₃ had no effect on either the calcium sensitivity or maximum calcium-activated force. Thus, there was no correlation between inotropic response to alpha stimulation and myocardial response to Ins(1,4,5)P₃. On the other hand, the maximum calcium-activated force of skinned skeletal muscle was slightly increased by Ins(1,4,5)P₃. Moreover, Ins(1,4,5)P₃ significantly increased the sensitivity of these fibers to calcium.     

Anion Dependence of the Contractions of skinned cardiac cells

Cardiac muscle fragments with disrupted sarcolemmas were prepared by homogenization of mouse ventricles. The rate of spontaneous contractions was increased when a solution containing isobutyrate as the main anion was substituted with a solution rich in chloride. At low calcium concentrations preparations which were quiescent in the isobutyrate solution responded to the chloride with a strong single contraction.This work was supported by the Deutsche Forschungsgemeinschaft, SFB 38 Membranforschung, Projekt G     

Contractions of skinned cardiac cells elicited by current pulses

Cardiac muscle fragments with disrupted sarcolemma were prepared by homogenization of mouse ventricles. The preparations exhibited spontaneous contractions of a rate between 3 min-1 and 12 min-1 at 20 degrees C, when they were kept in an appropriate solution. 2. Fragments of about cellular size were attached to two stiff glass microelectrodes, and additional contractions between the spontaneous beats were elicited by current pulses. The duration and intensity of the stimuli were varied to obtain strength-duration curves. Rheobase was in the range of 1.5 muA to 10 muA (no isolation of surrounding bath), chronaxia at 35 degrees C between 30 ms and 80 ms. 3. One microelectrode could be glued to a photodiode-force-transducer for simultaneous recording of contractions and electrical stimulation or potential measurement. Duration of phasic contractions was nearly 1s, force was up to 4muN (20 mN/mm2 of tension).     

Effect of temperature on the mechanical behaviour of single skinned cardiac cells

Summary The effect of changes in temperature (16–35° C) on the contractile behaviour of single, skinned cardiac cells was analysed. Lowering the temperature decreased force development, extent of shortening and velocity of shortening. The effects of altering temperature on the calcium sensitivity of velocity of shortening were more pronounced than the temperature effect upon the calcium dependence of force and extent of shortening. At maximal activation force-velocity curves showed a marked shift in peak velocity, with hardly any effect on peak isometric force.     

Effects of pH on spontaneous tension oscillation in skinned bovine cardiac muscle

Skinned cardiac muscle preparations exhibit spontaneous tension oscillations (spontaneous oscillatory contractions; SPOCs) in the absence of Ca²⁺, and in the presence of MgATP, MgADP and inorganic phosphate (Pi; ADP-SPOC). Similar oscillations occur in the presence of sub-micromolar concentrations of Ca²⁺ under normal activating conditions without MgADP and Pi (Ca-SPOC). In the study presented here, we investigated the effects of pH on both types of SPOC in skinned bovine cardiac ventricular muscle. First, a decrease in pH increased the MgADP concentration required to induce the half-maximal isometric tension that is obtained in the absence of Ca²⁺ and in the presence of MgATP (ADP-contraction). The inhibitory effect of Pi on ADP-contractions was not affected by pH. Second, ADP-SPOCs occurred upon the addition of Pi to the solution that resulted in ADP-contraction, and the relative amplitude and the period of the tension oscillation in the presence of 2 mM MgATP, 10 mM MgADP and 10 mM Pi were unchanged under all pH conditions examined (6.6, 7.0, 7.4). On the contrary, the relative amplitude and the period of the Ca-SPOCs were markedly diminished at pH 6.6. Finally, we constructed state diagrams showing the effects of pH on SPOC conditions. The state diagram shows that SPOCs occur less frequently under acidic conditions than at neutral pH. We suggest that the intermediate state of crossbridges that is required for SPOCs is more difficult to attain at a low pH. Received: 14 September 1998 / Received after revision: 23 February 1999 / Accepted: 10 March 1999     

Effects of ammonium ions on the depolarization-induced and direct activation of the contractile apparatus in mechanically skinned fast-twitch skeletal muscle fibres of the rat

Summary Mechanically skinned fibre preparations from the extensor digitorum longus muscle of the rat were used to test whether a rise in myoplasmic [NH₄ ⁺] in the range 2–10 mm interferes with the mechanism of excitation-contraction coupling in fast-twitch mammalian muscle. Under our conditions (pH 7.10, Mg²⁺ 1 mm, temperature 23° C), [NH₄ ⁺] up to 10 mm had little effect on the Ca⁺-activated force and on the peak of the t-system depolarization-induced force response. However, the duration of the depolarization-induced force response was decreased significantly at [NH₄ ⁺] 2 mm. From these data we conclude that the intracellular accumulation of NH₄ ⁺ is not likely to play a major role in fatigue. Nevertheless, the build up of NH₄ ⁺ during fatigue, may have a significant inhibitory effect on the force output by decreasing the duration of the t-system depolarization-induced activation of the contractile apparatus.     

Dependence upon high-energy phosphates of the effects of inorganic phosphate on contractile properties in chemically skinned rat cardiac fibres

The effects of inorganic phosphate (P_i) on mechanical properties of Triton X100 treated ventricular fibres have been studied in different substrate conditions. In the presence of both MgATP and phosphacreatine, increasing concentrations of P_i progressively decreased maximal active force, up to 50–60% at 20 mM P_i. The reduction in stiffness was slightly less. These effects appeared nearly independent of the diameter of the preparations. 20 mM P_i decreased Ca sensitivity of the myofilaments and increased the Hill coefficient of the tension/pCa relationship; furthermore, the time constant of tension recovery was decreased from 12.9 to 8.9 ms suggesting that the cycling rate of cross-bridges was increased in the presence of P_i. When MgATP was regenerated by the myofilament bound creatine kinase in the presence of phosphocreatine, P_i was less efficient in decreasing the maximal tension and it weakened the relaxing effect of MgATP upon rigor tension. These effects are related to the inhibition of creatine kinase by P_i. The effects of P_i on maximal force and kinetics of contraction were antagonized by the effects of a decrease in phosphocreatine. The results are discussed in terms of the antagonistic role of P_i increase and phosphocreatine decrease upon contractile properties of myofilaments during hypoxia in heart muscle.     

Sprint-training effects on some contractile properties of single skinned human muscle fibres

The effects of sprint training on the contractile properties of human muscle fibres obtained by needle biopsy were investigated. Individual fibres were mechanically skinned and activated by Ca²⁺- and Sr²⁺-buffered solutions at pH 7.1, and allocated to distinct populations on the basis of their contractile characteristics. The majority of fibres sampled pre-training could be separated into the three major fibre groups: Populations I (24/70, 34%), II (25/70, 36%) and III (18/70, 26%), which exhibited characteristics similar to those of histochemically classified type I, IIA and IIB fibres, respectively. The remainder (3/70, 4%) represented another fibre group, with intermediate characteristics. The muscle fibres were also activated by Ca²⁺ at a reduced pH of 6.6, to mimic the intracellular acidification that occurs during intense exercise. Lowering pH increased the threshold for contraction by Ca²⁺, reduced Ca²⁺ sensitivity, and increased the steepness of the force-pCa relationship, in all fibres sampled from the three major fibre groups. Maximum force was not significantly reduced in any fibre population. In the post-training sample, the three major fibre types were present in different proportions: Populations I (10/52, 19%), II (20/52, 38.5%) and III (11/52, 21%). Three other fibre groups sampled in low numbers exhibited contractile characteristics intermediate between Population I and Population II. Following sprint training all of the three main fibre populations exhibited higher thresholds for contraction by, and lower sensitivities to, Sr²⁺ but not Ca²⁺, compared with the fibres sampled pre-training. Maximum force was significantly lower in Population II fibres after sprint training. At pH 6.6, post-trained Population III fibres exhibited even lower Ca²⁺ sensitivity, with concomitant increases in the threshold for contraction and force-pCa curve steepness.     

Effects of activation rate on contractile properties of rabbit masseter motor units

Force-frequency curves of rabbit masseter motor units ( n=20) were determined, in order to study the capacity of these motor units for rate gradation and to establish the relationship between twitch contraction time (TCT) and the shape of the curves. Motor unit force responses were elicited by stimulating motoneurons in the trigeminal motor nucleus extracellularly. A sequence of pulse trains with increasing frequency rates was followed by trains with decreasing frequency rates. All motor units were classified as fast (F) units. The ascending force-frequency curves showed a distinct sigmoid appearance; the descending curves were shifted toward lower stimulation rates. The position and shape of the force-frequency curves related significantly to the TCT. The curves of slower units were located at lower frequencies and had a larger inclination. In addition, slower units had a lower fusion frequency and a larger twitch-tetanus ratio. Hence, slower units started to fuse and reached maximum force at lower stimulation rates than faster units and needed a smaller change in simulation frequency to achieve the same relative force. It can be concluded that the capacity for rate gradation differs between rabbit masseter motor units and that the TCT is a determinant for the position and shape of the force-frequency curves.     

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.     

A study of the short-term effects of glucose 6-phosphate on the contractile activation properties of skinned single muscle fibres of the rat. Implications for solution design

The present study highlights possible problems that can arise from the incorrect preparation of control and test solutions for use in Ca2+-activation experiments using single skinned skeletal muscle fibres and EGTA-based Ca2+ buffers. We show here, using glucose 6-phosphate (G6-P) as our "test" compound, that the Ca2+-activation properties of skinned single fibre segments from the extensor digitorum longus muscle of the rat are highly dependent on the form in which the G6-P is added and on the correct balancing of an appropriate anion in control solutions. Test solutions prepared by the direct addition of 10 mM monosodium G6-P salt to a set of control solutions of defined pCa resulted in significantly greater submaximal force responses than the corresponding controls. This is equivalent to an increase in the sensitivity of the contractile-regulatory system to Ca2+ (pCa50=-log10[Ca2+] that produces 50% of maximum force) by 0.19+/-0.01 pCa units. In contrast, addition of disodium G6-P to control solutions caused a slight reduction in the apparent sensitivity of the contractile apparatus to Ca2+ by 0.04+/-0.01 pCa units (P<0.01). Rather than being indicative of the effects of G6-P on the contractile apparatus, these opposing effects are due to differences between test and control solutions with respect to pH and Na+ concentration brought about by the G6-P salts. When all ionic species were carefully balanced, 10 mM G6-P was found to have only a small sensitizing effect on Ca2+-activation properties compared to control, without affecting the maximum Ca2+-activated force response. Our findings highlight the often-overlooked need for careful balancing of the ionic composition in control and test solutions when examining the true effects of different compounds on the Ca2+-activation characteristics of single skinned muscle fibre preparations.     

Contractions induced by a calcium-triggered release of calcium from the sarcoplasmic reticulum of single skinned cardiac cells.

1. Fragments of single cardiac cells were obtained by homogenization of ventricular tissue from adult rats. Remaining pieces of sacrolemma were removed by micro-dissection. Tension was recorded from the ends of the skinned (sarcolemma-free) cells with a photodiode force transducer. 2. In the presence of a strong buffering of the free [Ca2+] with 4-0 mM total EGTA, a tonic tension was obtained that increased according to t sigmoid curve when the free ([Ca2+] was increased from 10(-6-75)M to 10(-5-0)M. This curve was not modified by the destruction of the sarcoplasmic reticulum (SR) by the detergent Brij 58. Therefore, the tonic tension corresponded to the direct effect of the free [Ca2+] present in the buffer on the myofilaments. 3. In the presence of a slight buffering of the free [Ca2+] with 0-050 mM total EGTA, cyclic contractions were observed that were attributed to cyclic releases and re-sequestrations of Ca2+ by the SR. The absence of effect of azide and ruthenium red on the cyclic contractions obtained at a free [Ca2+] lower than 10(-6-50)M demonstrated that the mitochondria played no role in the triggering of these contractions. 4. Cyclic contractions were induced by a slight variation of free [Ca2+] in the buffer from 10(-7-65)M to 10(-7-40)M. Their amplitude at 10(-7-40)M free Ca2+ was equal to the tonic tension developed by a free [Ca2+] 20 times higher applied to the myofilaments when the SR was destroyed by detergent or functionally inhibited by high total [EGTA]. It was concluded that these cyclic contractions corresponded to a Ca2+-triggered release of Ca2+ from the SR. 5. The cyclic contractions were induced by the filling of the SR with Ca2+ to a critical level at which it released a fraction of the Ca2+ it contained. Each contraction was followed by a re-sequestration of Ca2+, the kinetics of which conditioned the duration of the cycles. 6. The amplitude of the cyclic contractions increased when the free [Ca2+] that triggered them was increased. This gradation was deemed incompatible with a simple regenerative process, which should produce an all-or-nothing response. Additional process, such as a modulation of the Ca2+ release by free [Mg2+] and [ADP] may help to explain the gradation of the contractions. 7. It was concluded that a Ca2+-triggered release of Ca2+ from the SR of rat ventricular cells may amplify the Ca2+ flux crossing the sarcolemma during the plateau of the action potential, thereby permitting the activation of the myofilaments.     

Effects of caffeine on Ca-activated force production in skinned cardiac and skeletal muscle fibres of the rat

Skinned fibres prepared by mechanical and/or chemical means from cardiac and skeletal muscles of the rat were activated in solution strongly buffered for Ca²⁺ (with 50 mM EGTA) in the absence or presence of caffeine 5–40 mM. In all preparations caffeine was found to reversibly shift the relation between steady-state force and free [Ca²⁺] toward lower free [Ca²⁺] in a dose dependent manner. This increase in apparent Ca²⁺ sensitivity was not antagonized by procaine and was the same, within each muscle type, irrespective of the manner in which the skinned fibre was prepared, and consequently the degree to which it retained cellular membranes. The effect was more pronounced in cardiac and slow-twitch than in fast-twitch, myofibrillar preparations. At equivalent concentrations theophylline mimicked this effect of caffeine in all preparations, however, addition of exogenous cyclic AMP neither mimicked nor modified, in any way, the effect. Maximum Ca²⁺-activated force production was not affected by caffeine below 20 mM but was depressed by concentrations of 20 mM and above. The increase in apparent Ca²⁺ sensitivity produced by caffeine can not be the result of a mobilization of some cellular store of Ca²⁺ but must arise from a direct effect of caffeine on the myofilaments which leads to a change in the apparent affinity constant of the force controlling sites for Ca²⁺.     

Tension in mechanically disrupted mammalian cardiac cells: effects of magnesium adenosine triphosphate.

1. Maximum and submaximum Ca-activated tension in mechanically disrupted rat ventricular fibres was examined in solutions containing 30 micron, 100 micron and 4 mM-MgATP and either 50 micron or 1 mM ionized Mg. 2. In the absence of added Ca, significant amounts of base-line tension (up to 50% of maximum) develop in solutions containing less than 30 micron-MgATP. This effect is Mg-dependent; more tension is produced with 50 micron-Mg than with 1 mM. 3. Increasing the MgATP concentration shifts the pCa-% maximum tension relationship in the direction of increasing Ca required for activation. At 50 micron-Mg the pCa which produces 50% maximum tension is 5-8, 5-3 and 5-5 for the 30 micron, 100 micron and 4 mM-MgATP solutions. The effect of MgATP on position is relatively independent of the Mg concentration. 4. The steepness of the pCa-% maximum tension curve increases as MgATP is elevated to the millimolar range. The Hill coefficients for the different MgATP curves at 50 micron-Mg are 1-1, 1-3 and 3-0. This change in steepness accounts for the slightly lower Ca concentration needed for half-maximum tension as the MgATP concentration is increased to millimolar levels. Raising the Mg concentration to 1 mM greatly diminishes the effect of MgATP on the slope of the pCa-tension relationship. 5. The maximum tnesion a fibre bundle can produce decreases as the amount of MgATP is raised from micromolar to millimolar levels. For 50 muM-Mg, maximum tension drops about 35% as MgATP is raised from 30 micronM to 4 mM. For any concentraiton of MgATP, maximum tension is higher at 1 mM-Mg than at 50 micron-Mg. 6. Existing theories of interaction between myosin heads and the thin filament are sufficient to account for the effects of MgATP on the position of the pCa-tension curves and on maximum tension. The effects on slope are less satisfactorily explained.     

The thiadiazinone EMD 57033 speeds the activation of skinned cardiac muscle produced by the photolysis of nitr-5

EMD 57033 is thought to produce its potentiating effect by increasing the apparent Ca²⁺ sensitivity of the myofilaments, possibly by altering the kinetics of actomyosin interaction. We have investigated the effect of 10M EMD 57033 upon activation speed, induced by flash photolysis of 2mM nitr-5 (caged Ca²⁺), in chemically skinned trabeculae from the guinea-pig at 12°C. EMD 57033 increases the half time of activation from 238±18.5 msec (n=6) to 132.1± 34.0 msec (n=8)(mean ± s.e.m.) and suggests that this Ca²⁺ sensitiser has an important effect upon f_app, that is the transition from the non-force to force generating cross-bridge states.     

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.     

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.     

Endurance exercise effects on the contractile properties of single,skinned skeletal muscle fibres of young rats

Single fibres were isolated from the extensor digitorum longus (EDL) and the soleus (SOL) muscles of the hindlimb from young male Wistar rats which had undergone a 10-week programme of endurance swimming from the age of 2 weeks. Fibres were mechanically skinned and activated with Ca²⁺- and Sr²⁺ -buffered solutions. Muscle fibres were classified by means of well-defined criteria concerning various aspects of their contractile behaviour. Most fibres could be allocated into specific groups; however, a significant proportion (13% of the sampled population) did not fit these rigid classifications but displayed contractile activation characteristics common to more than one fibre type. In these cases models which used a combination of both fast- and slow-twitch contractile and regulatory properties were used to characterise the activation behaviour of fibres. It is proposed that the exercise, initiated at a young age, induced changes in the contractile characteristics of the single fibres by modifying protein isoforms of the contractile apparatus.