Rate of isometric tension development in relation to calcium binding of skinned muscle fibres

Isolated muscle fibres from the sartorius or semitendinosus muscles of frogs were mechanically skinned and kept in a relaxed state in a medium containing Mg-ATP and EGTA. When subjected to a rapid increase in internal calcium ion concentration tension rose relatively slowly in comparison to the time course of establishment of the new calcium concentration. Stiffness measurements made during the rise of tension yielded the same stiffness to tension ratio as that observed at steady state force. The linear force extension curve of the activated fibres (T₁-curves) measured at various moments during the rise of tension extrapolated to zero tension intersected the base line at the same length (–0.8% L_o). This suggests that the extent of myosin interaction increases with the same time course as tension.The rate of tension development accompanying a Ca-jump was strongly increased by an increase in calcium ion concentration and there was a linear relationship between the logarithm of the rate tension development and pCa. The rate of recovery of tension following a large quick release > 2% L_o was not calcium sensitive, and occurred at a rate more than an order of magnitude faster than the corresponding calcium activation in the range of pCa's studied. We suggest that the slowness of tension development accompanying a rapid calcium activation reflects slow reactions occurring after a single Ca-ion has bound to a myofilament binding site and does not reflect the slowness of actin and myosin interaction.This work was supported by a grant from the Deutsche Forschungs-gemeinschaft Gu 160/3. One of us (P.J.G.) was supported by the Royal Society European Science Exchange Programme     

Cross-bridge stiffness in Ca2+-activated skinned single muscle fibres

Tension transients, in response to small and rapid length changes (completed within 40 s), were obtained from skinned single frog muscle fibres incubated in activating solutions with varying concentrations of Ca²⁺. The first 2 ms of these transients were described by a linear model in which the fibre is regarded as a rod composed of infinitesimally small, identical segments containing a mass, one undamped elastic element and in the case of relaxed fibres two damped elastic elements in series, or in the case of activated fibres three such elastic elements in series. The stiffness of activated fibres, expressed in elastic constants or apparent elastic constants, increased with increasing concentrations of Ca²⁺. All the damped elastic constants that were necessary to describe the tension responses of activated fibres were proportional to isometric tension. However, the undamped elastic constant did not increase linearly with increasing isometric tension. Equatorial X-ray diffraction patterns were obtained from single frog muscle fibres under similar conditions as under which the tension transients were obtained. The filament spacing (d ₁₀)of Ca²⁺-activated single frog muscle fibres decreased with increasing isometric force, whereas the intensity ratio (I ₁₁/I₁₀)increased linearly with increasing isometric force. From experiments in which dextran (MW 200000 Da) was added, it followed that such a change in filament spacing would modify passive stiffness. The d ₁₀value of relaxed fibres decreased and stiffness increased with increasing concentrations of the polymer dextran, whereas I ₁₁/I₁₀remained constant. The relation of stiffness and filament spacing with concentration of dextran was used to eliminate the effect of decreased filament spacing on stiffness of activated fibres. After correction for changes in filament spacing the undamped complicance C ₁, normalized to tension, was not constant, but increased with increasing isometric tension. If we assume that isometric tension is proportional to the number of force generating cross-bridges, this means that only part of the undamped complicance of activated fibres is located in the crossbridges.     

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.     

Inhibition of TnI-TnC interaction and contraction of skinned muscle fibres by the synthetic peptide TnI [104–115]

Circular dichroism was used to study the induction of helix in TnC or TnI-TnC by the TnI peptide [104–115] at various Ca²⁺ concentrations. The increase in negative ellipticity and pCa²⁺ values for the peptide-TnC complex, indicates that binding of the peptide to TnC, induces a small helical conformational change in TnC. This results in an increase in the Ca²⁺ binding constant and the pCa₅₀ value required to induce 50% of Ca²⁺-dependent helix in TnC. The introduction of the peptide to a preformed mixture of TnI-TnC resulted in an increase in negative ellipticity and a decrease in the pCa₅₀ and the apparent Ca²⁺ binding constant towards the values obtained for the TnI peptide-TnC complex and away from those of TnI-TnC. This demonstrates that the TnI peptide can successfully compete with TnI for TnC and thereby inhibit the TnI-TnC interaction. The addition of the TnI peptide to skinned rabbit psoas or porcine cardiac fibres resulted in the inhibition of the force development and a decrease in the pCa₅₀ values required for 50% Ca²⁺ activation. The magnitude of the inhibition of tension development and the shift in the Ca²⁺ sensitivity for skinned cardiac muscle fibres was approximately half that observed with skeletal muscle fibres. In view of the CD findings, these skinned fibre results can be accounted for by the peptide inhibiting the TnI interaction with TnC. However, it is possible that the TnI peptide also has a direct inhibitory effect on TM-actin. Mastoparan, another TnC binding peptide, also inhibited the tension development in skinned skeletal and cardiac muscle fibres, but was much less efficient than the TnI peptide.Abbreviations used Boc N-tert-butyloxycarbonyl - Tn troponin - TM tropomyosin - TnI troponin I - TnC troponin C - HPLC high performance liquid chromatography - DIEA diisopropylethylamine - TFA trifluoroacetic acid - DCC dicyclohexylcarbodiimide - 2-Cl-Z 2-chlorobenzyloxycarbonyl - Tos 4-toluenesulfonyl - EGTA ethylene glycol bis(-amino ethyl ether) N,N,N,N-tetraacctic acid - DTE 1,2-ethanedithiol - ME -mercaptoethanol - S1 myosin subfragment 1 - acto-S1 actin and myosin subfragment This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie, and by grants from the Medical Research Council of Canada, the Alberta Heart Foundation and a studentship (J. V. E.) from the Alberta Heritage Foundation for Medical Research     

Alteration of calcium sensitivity of skinned frog skeletal muscle fibres by inositol triphosphate and calmodulin antagonists

We investigated the influence of inositol triphosphate (IP₃), trifluoperazine (TFP), and perhexiline on the calcium sensitivity of freeze-dried frog semitendinosus muscle fibres. Further, the effect of IP₃ on calcium release from the sarcoplasmic reticulum (SR) of frog semitendinosus fibres skinned by saponin was studied. IP₃ decreased the calcium sensitivity of freeze-dried frog skeletal muscle fibres and failed to induce a calcium release from SR of saponin-skinned fibres. Freeze-dried frog skeletal muscle fibres were strongly sensitized for calcium by TFP and perhexiline.     

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.     

Transmission phenomena and early tension recovery in skinned muscle fibres of the frog

Tension responses due to small rapid length changes completed in 50 s were obtained from segments with different length of single fibres of the ileofibularis muscle of the frog. The very early parts of the responses varied with segment length. A simulation of the early parts of the response was carried out by means of a linear model in which the fibre is regarded as a rod of infinitesimally small segments containing undamped elasticity, damped elasticity and mass in series. In the simulation corrections were included for the effects caused by the viscosity and density of the surrounding fluid and for the force transducer characteristics. The results indicate the presence of a very rapid component in the fast recovery with a time constant of 5–15 s. The undamped elasticity of the activated fibres corrected for their passive properties was such that a sudden shortening corresponding to 2.6 nm/half sarcomere would reduce active tension to zero.     

Recombinant troponin I substitution and calcium responsiveness in skinned cardiac muscle

Using treatment with vanadate solutions, we extracted native cardiac troponin I and troponin C (cTnI and cTnC) from skinned fibers of porcine right ventricles. These proteins were replaced by exogenously supplied TnI and TnC isoforms, thereby restoring Ca²⁺-dependent regulation. Force then depended on the negative logarithm of Ca²⁺ concentration (pCa) in a sigmoidal manner, the pCa for 50% force development, pCa₅₀, being about 5.5. For reconstitution we used fast-twitch rabbit skeletal muscle TnI and TnC (sTnI and sTnC), bovine cTnI and cTnC or recombinant sTnIs that were altered by site-directed mutagenesis. Incubation with TnI inhibited isometric tension in TnI-extracted fibers in the absence of Ca²⁺, but restoration of Ca²⁺ dependence required incubation with both TnI and TnC. Relaxation at low Ca²⁺ levels and the steepness of the force/pCa relation depended on the concentration of exogenously supplied TnI in the reconstitution solution (range 20–150 μM), while Ca²⁺ sensitivity, i.e. the pCa₅₀, was dependent on the isoform, and also on the concentration of TnC in the reconstitution solution. At pH 6.7, skinned fibers reconstituted with optimal concentrations of sTnC and sTnI (120 μM and 150 μM, respectively) were more sensitive to Ca²⁺ than those reconstituted with cTnC and cTnI (difference in pCa₅₀ approx. 0.2 units). Rabbit sTnI was cloned and expressed in Escherichia coli using a high yield expression plasmid. We introduced point mutations into the TnI inhibitory region comprising the sequence of the minimal common TnC/actin binding site (-G¹⁰⁴-K-F-K-R-P-P-L-R-R-V-R¹¹⁵-). The four mutants produced by substitution of T for P¹¹⁰, G for P¹¹⁰, G for L¹¹¹, and G for K¹⁰⁵ were chosen, based on previous work with synthetic peptides showing that single amino acid substitution in this region diminished the capacity of these peptides to inhibit acto-S₁ ATPase or contraction of skinned fibers. Therefore, all amino acid residues of the inhibitory region are thought to contribute to biological activity of TnI. However, each of the recombinant TnIs could substitute for endogenous TnI. In combination with exogenous TnC, Ca²⁺ dependence could be restored when ^gly110sTnI, ^thr110sTnI or ^gly111sTnI was used for reconstitution. The mutant ^gly105sTnI, on the other hand, reduced the ability of skinned fibers to relax at low Ca²⁺ concentrations and it caused an increase in Ca²⁺ sensitivity. Received: 5 October 1995/Received after revision and accepted: 1 December 1995     

Force and force transients in skeletal muscle fibres of the frog skinned by freeze-drying

A freeze-drying method is described by which single skinned skeletal muscle fibres or fibre bundles can readily be obtained. Skinned fibre segments of the ileofibularis and semitendinous muscles of the frog — activated by means of a rapid increase in the Ca-concentration — showed very stable and reproducible contractions. Complete activation occurred at a Ca-concentration of 1.6·10^–6 M and the mid-point of the pCa-tension curve occurred at 6.3·10^–7 M. Addition of phosphate (10^–2 M) had a depressing effect on the speed of the Ca-activated tension development as well as on the maximum tension reached.Addition of caffeine (10^–2 M) had no effect on the tension generation, indicating that the sarcoplasmic reticulum, if present, was not active. The force responses due to rapid length changes applied to the Ca-activated fibre preparations were found to be qualitatively similar to the force responses on intact tissue. This skinning technique might be employed on human biopsies, enabling the measurement of physiological parameters such as for example force and shortening velocity.     

Tension responses to rapid length changes in skinned muscle fibres of the frog

Tension responses due to rapid length changes completed in 50 and 150 s were obtained from activated skinned single fibres of the ileofibularis muscle of the frog. The natural frequency of the force transducer was about 50 kHz. The length changes ranged between –1% and +0.5% of the fibre segment length. The sarcomere length was adjusted to 2.15 m. The temperature was maintained at 2.5° C. The transmission velocity estimated from these recordings obtained on fibre segments with different length was 230 m/s in fully activated segments and 112 m/s in relaxed segments. The initial part of the responses during the length changes consisted of an abrupt change in tension reaching an extreme value T₁, which depended on the amplitude as well as the duration of the length change. A partial rapid recovery towards a plateau occurred after the length change. The reciprocal half-time of this recovery increased with the amplitude of the displacement both for stretches as well as releases up to about 5 nm/half sarcomere.     

Effects of thapsigargin and cyclopiazonic acid on the sarcoplasmic reticulum Ca2+ pump of skinned fibres from frog skeletal muscle

Thapsigargin has been reported to inhibit ATP-dependent Ca²⁺ uptake by isolated sarcoplasmic reticulum (SR) vesicles of vertebrate skeletal muscle fibres at nanomolar concentrations. There have been no reports confirming this effect in skinned muscle fibre preparations. We have examined the ability of thapsigargin to inhibit the uptake of Ca²⁺ by the SR in mechanically skinned fibres of frog iliofibularis muscles, using the size of the caffeine-induced contracture to assess the Ca²⁺ content of the SR. The SR was first depleted of Ca²⁺ and then reloaded for 1 min at pCa 6.2 in the presence and absence of thapsigargin. When 5 min were allowed for diffusion, a thapsigargin concentration of at least 131 M was required to inhibit Ca²⁺ loading by 50%. In contrast, another SR Ca²⁺ uptake inhibitor, cyclopiazonic acid, was more effective, producing 50% inhibition at 7.0 M and total inhibition at 50 M. When cyclopiazonic acid (100 M) was applied after, rather than during, Ca²⁺ loading, the caffeine-induced contracture was not changed. Thapsigargin (300 M), on the other hand, caused some reduction in the peak amplitude of the caffeine-induced contracture when applied after Ca²⁺ loading. The poor effectiveness of thapsigargin in the skinned fibres, compared with in SR vesicles, is attributed to its slow diffusion into the skinned fibres, perhaps as a result of binding to myofibrillar components.     

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.     

Shortening induced deactivation of skinned fibres of frog and mouse striated muscle

The depressant effect of active shortening, previously established in intact muscle fibres, was studied during calcium induced contractures of chemically skinned fibres from the semitendinosus muscle of Rana temporaria and the psoas muscle of the mouse. The decrease in contractile activity was determined by comparing the rate of force redevelopment (at a given tension level) after a large (test) and a small (control) release step. Under standard experimental conditions (ionic strength: frog 135 mM, mouse 190 mM; Ca²⁺ 3.0 μM; Mg²⁺: frog 25 μM, mouse 100 μM; MgATP^2-: frog 1.0 mM, mouse 2.0 mM) active shortening of 0.15 μm per sarcomere (in excess of control release) reduced the contractile activity by approximately 50% of the control in both frog and mouse muscle fibres. Full contractile activity was regained within 4 s during isometric activity after the shortening phase. The depressant effect of shortening was steadily reduced, to almost complete disappearance of the effect, by increasing the free calcium concentration within the range 1.5–12.0 μM. Similarly, an increase in ionic strength from 105 to 235 mM reduced the depressant effect by approximately 40%. In contrast, there was a progressive enhancement of the shortening effect as the magnesium ion concentration was increased from 25 to 590 μM. It is proposed that interaction between the myosin cross-bridges and the thin filament during sarcomere shortening leads to a decrease in troponin-calcium binding resulting in a temporary deactivation of the contractile system.     

Calcium release modulated by inositol trisphosphate in ruptured fibers from frog skeletal muscle

To investigate the effect of inositol 1,4,5-trisphosphate on calcium release, we used fiber bundles of frog sartorius muscle mechanically permeabilized by a scratching procedure, and we detected increments in calcium concentration by measuring aqueorin light signals. Submicromolar concentrations of inositol 1,4,5-trisphosphate induced fast calcium-release signals, with a half time to peak of 60 ms or less. Similar responses were elicited by caffeine. The calcium-release signal induced by inositol 1,4,5-trisphosphate occurred at pCa values of 7 or lower, and the dose-response curve depended on the ionic composition of the incubation solution. Lower inositol 1,4,5-trisphosphate concentrations were needed to induce release when incubation solutions of ionic composition expected to depolarize the transverse tubule membrane were used. Inositol 1,4,5-trisphosphate was more effective than inositol 1,3,4-trisphosphate, inositol 1,4,5,6-tetrakisphosphate, and inositol 1,4-bisphosphate. The effect of inositol 1,4,5-trisphosphate was synergistic with that of caffeine, and was not inhibited by heparin. These results, by showing directly that at resting calcium levels inositol 1,4,5-trisphosphate elicited calcium release, are consistent with a role for inositol 1,4,5-trisphosphate as a chemical modulator in excitation/contraction coupling in skeletal muscle.     

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.     

Cyclic-AMP mediated relaxation of chemically skinned fibres of smooth muscle

Smooth muscle from guinea pig taenia coli was chemically skinned with Triton X-100 and stored in ATP-salt solution containing 50% glycerol at –20°C. Fibre bundles were relaxed at Ca²⁺-concentrations below 10^–7 M, but contracted at 10^–6 M Ca²⁺. The isometric tension developed could be partly relaxed by the addition of c-AMP (in the presence of NaF), and it could also be inhibited following preincubation with the catalytic subunit of c-AMP dependent protein kinase. The inhibitory effect was much more pronounced at intermediate Ca²⁺-concentrations (e.g. 10^–6) than at concentrations producing a maximum contraction, suggesting that Ca-sensitivity had been lowered. Sodium fluoride which was required to potentiate the c-AMP effects was found to have a slight relaxing effect per se. The c-AMP effect may be mediated through activation of cyclic AMP-dependent kinase, producing phosphorylation of the myosin light chain kinase which, according to adelstein et al. (1978), may result in a net dephosphorylation of the myosin light chains and a concomittant inhibition of the contractile response.     

The influence of ionic strength upon relaxation from rigor induced by flash photolysis of caged-ATP in skinned murine skeletal muscle fibres

The influence of ionic strength upon relaxation kinetics from rigor in skinned murine extensor digitorum longus (EDL) skeletal muscle fibres was examined using photolysis of caged-ATP at low Ca²⁺. The ionic strength was adjusted with either KMeSO₃ or ethylene glycol bis-(-aminoethyl ether)N,N,N,N-tetraacetic acid, dipotassium salt (K₂EGTA) in the range of /2=65–215 mM, or I.E. 49–194 mM, where I.E. denotes ionic equivalent. Following rigor development at a/2 of 165–215 mM (I.E. 144–194 mM), the liberation of approximately 0.5 mM ATP resulted in an initial 6-to 10-ms detachment phase with a decline in force of approximately 10–20% followed by a 10-to 30-ms reattachment with up to a 60% increase compared to the corresponding rigor level and a final detachment leading to complete relaxation. Interestingly, when similar ATP concentrations were liberated at lower ionic strengths between a /2 of 65 mM and 110 mM (I.E. 60–100 mM), the initial detachment phase was shortened and force decreased by only approximately 5–10%, while the following reattachment phase was lengthened and led to an increased steady-state force of approximately 20–80% without final relaxation. ATP-induced detachment and subsequent reattachment were mainly determined by the currently present ionic strength and were relatively independent of the preceding rigor state which had been developed at higher or lower ionic strengths. The effects of phosphate and apyrase on the force transient suggest that reattachment of ADP- binding crossbridges may contribute to the increase in tension at high and even more at low ionic strengths. The study shows that the kinetics of initial fast relaxation and subsequent redevelopment of force following flash photolysis of similar ATP concentrations are markedly modified by the ionic strength in the narrow range of between 65 mM and 215 mM.     

Calcium sensitivity and unloaded shortening velocity of hypertrophied and non-hypertrophied skinned human atrial fibres

The mechanical properties of myocardium of different animals are modified by a chronic increase in haemodynamic load. In this study differences in calcium sensitivity and maximum unloaded shortening velocity of hypertrophic and non-hypertrophic chemically skinned human atrial fibres are characterized. Investigating right atria of 34 patients, possible correlations are studied between preoperative atrial pressure, degree of hypertrophy (estimated from the muscle fibre diameter), calcium responsiveness (pCa₅₀ eliciting half-maximum contraction) and V _max (unloaded shortening velocity). Hypertrophic fibres from atrial appendages of patients having an increased right atrial pressure (RAP 8.5±1.6 mm Hg) and suffering from mitral valve disease (stenosis and insufficiency combined) had a fibre diameter of 18.0±0.9 m. They also had a higher calcium sensitivity (pCa₅₀ 5.65±0.08) and a lower unloaded shortening velocity (1.7±0.1 muscle lengths/s) than non-hypertrophic fibres from the appendages of patients with normal right atrial pressure (RAP 3.2±0.5 mm Hg) and coronary heart disease (CHD: pCa₅₀ 5.45±0.04; V _max= 3.4±0.2 muscle lengths/s; fibre diameter 12.8±0.4 m). Thus non-hypertrophic fibres from control CHD patients differed significantly (p < 0.01) from hypertrophied atrial fibres of patients with mitral valve disease and with combined valve disease (MAV, pCa₅₀=5.58±0.05, V _max 2.0±0.3 muscle lengths/s, fibre diameter 14.6±0.9 m) or aortic valve disease (stenosis combined with insufficiency, fibre diameter 14.8±1.4 m, pCa₅₀ 5.56±0.03, V _max 2.0±0.24 muscle lengths/s; RAP 11.0±2.6 mm Hg). Such alterations of calcium responsiveness, shortening velocity and fibre thickness may reflect an adaptation to the chronic overload in atria from patients with various forms of heart valve disease.     

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.     

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.