PubMed Health⌕ Search

Biomedical subjects

D G Stephenson

Publications and source records attributed to D G Stephenson.

At least 73 records · Page 4Linked to original sources
PubMed1994

Effects of heparin on excitation-contraction coupling in skeletal muscle toad and rat.

1. Intracellularly applied heparin was found to cause a novel, use-dependent block of excitation-contraction (E-C) coupling in skinned skeletal muscle fibres of the toad. After one to four depolarizations in the presence of 100 micrograms ml-1 heparin, no further depolarization-induced responses could be elicited, even though addition of caffeine or lowering [Mg2+] could still induce massive Ca2+ release. This effect could not be reversed by extensive wash-out of the heparin (> 15 min). 2. Heparin (100 micrograms ml-1) did not abolish subsequent depolarization-induced responses if applied while the voltage sensors were in either their resting or inactivated states, that is (a) while a fibre remained fully polarized, (b) when a fibre was already chronically depolarized or (c) after a fibre had been depolarized in the presence of D600 (gallopamil) and then repolarized. 3. When a toad fibre was depolarized in heparin, with the associated Ca2+ release blocked by the presence of 10 mM intracellular Mg2+, subsequent E-C coupling was abolished. Heparin did not interrupt E-C coupling when Ca2+ release was triggered in the absence of any depolarization, by either caffeine or low [Mg2+]. Thus, the opening of the Ca2+ release channels was neither necessary nor sufficient for heparin to abolish E-C coupling. 4. Heparin had direct effects on the contractile apparatus in toad fibres, increasing the Ca2+ sensitivity and decreasing the maximum Ca(2+)-activated force. These effects could only be partly reversed by extensive wash-out of heparin. 5. At 100 micrograms ml-1, both low molecular weight heparin and pentosanpolysulphate, another highly sulphated polysaccharide, were less effective than heparin in blocking the depolarization-induced response and in changing the properties of the contractile apparatus, and these effects could be substantially reversed by wash-out. Two other polyanions, de-N-sulphated heparin (100 micrograms ml-1), which lacked N-sulphate groups, and polyglutamate (500 micrograms ml-1), had no measurable effect on either E-C coupling or the contractile apparatus. 6. In skinned fibres of the extensor digitorum longus muscle of the rat, 100 micrograms ml-1 heparin had little or no effect on E-C coupling and on the Ca2+ sensitivity of the contractile apparatus, but caused a larger reduction of the maximum Ca(2+)-activated force than in skinned fibres of the toad. 7. These results indicate that heparin blocks E-C coupling in toad muscle if, and only if, it is present when the voltage sensors are activated by depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Theory and observation of spontaneous oscillatory contractions in skeletal myofibrils.

At low levels of activation, an isometrically-held myofibrillar preparation on the descending limb may exhibit persistent oscillations of period 1-6 s in tension and sarcomere lengths. We propose a sarcomeric theory of spontaneous oscillatory contraction, based on the phenomena of force creep and delayed length activation. The time delay leads to oscillations and controls their period. A computer model using these ideas simulates spontaneous oscillatory contraction for fixed-end fibres only if isometric tension capacity varies slightly along the fibre. The form of this inhomogeneity controls a diversity of spontaneous oscillatory contraction behaviour: the tension waveform can vary from large and sinusoidal to small-amplitude pulses or chaotic behaviour, and these variations are observed in slow-twitch soleus fibres from the same animal (rat). The model predicts that oscillatory and quiescent regions coexist in the fibre, with large-amplitude sawtooth waveforms in sarcomere length in the former as observed. It can also generate travelling-wave structures, similar to those found by the Tokyo group, in oscillating regions when there is a spatial gradient in isometric tension capacity. Phase discontinuities in sarcomere length occur near the oscillatory-quiescent boundary. Predictions for the Ca2+ concentrations and sarcomere lengths in which spontaneous oscillatory contraction occurs and for differences in the spontaneous oscillatory contraction frequencies of fast- and slow-twitch fibres compare well with experiment. Spontaneous oscillatory contraction is also predicted under isotonic conditions.

Animals↗
PubMed1994

Effects of Mg2+ on Ca2+ handling by the sarcoplasmic reticulum in skinned skeletal and cardiac muscle fibres.

The influence of myoplasmic Mg2+ (0.05-10 mM) on Ca2+ accumulation (net Ca2+ flux) and Ca2+ uptake (pump-driven Ca2+ influx) by the intact sarcoplasmic reticulum (SR) was studied in skinned fibres from the toad iliofibularis muscle (twitch portion), rat extensor digitorum longus (EDL) muscle (fast twitch), rat soleus muscle (slow twitch) and rat cardiac trabeculae. Ca2+ accumulation was optimal between 1 and 3 mM Mg2+ in toad fibres and reached a plateau between 1 and 10 mM Mg2+ in the rat EDL fibres and between 3 and 10 mM Mg2+ in the rat cardiac fibres. In soleus fibres, optimal Ca2+ accumulation occurred at 10 mM Mg2+. The same trend was obtained with all preparations at 0.3 and 1 microM Ca2+. Experiments with 2,5-di-(tert-butyl)-1,4-benzohydroquinone, a specific inhibitor of the Ca2+ pump, revealed a marked Ca2+ efflux from the SR of toad iliofibularis fibres in the presence of 0.2 microM Ca2+ and 1 mM Mg2+. Further experiments indicated that the SR Ca2+ leak could be blocked by 10 microM ruthenium red without affecting the SR Ca2+ pump and this allowed separation between SR Ca2+ uptake and SR Ca2+ accumulation. At 0.3 microM Ca2+, Ca2+ uptake was optimal with 1 mM Mg2+ in the toad iliofibularis and rat EDL fibres and between 1 and 10 mM Mg2+ in the rat soleus and trabeculae preparations. At higher [Ca2+] (1 microM), Ca2+ uptake was optimal with 1 mM Mg2+ in the iliofibularis fibres and between 1 and 3 mM Mg2+ in the EDL fibres.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Single-fiber study of contractile and biochemical properties of skeletal muscles in streptozotocin-induced diabetic rats.

Mechanically skinned, single muscle fibers, isometrically activated in pH and Ca2+ (Sr2+) buffered solutions were used to examine the function of the contractile apparatus in slow- and fast-twitch fibers from soleus (SOL, predominantly slow-twitch) and extensor digitorum longus (EDL, predominantly fast-twitch) muscles of streptozotocin (STZ)-induced diabetic rats and age-matched controls. Three and 14 days after STZ administration, the contractile properties of muscle fibers from diabetic rats did not differ significantly from those of controls with respect to several mechanical parameters, such as maximum Ca-activated tension, activation threshold, and sensitivity to Ca2+ and Sr2+. In contrast, 28 days after STZ administration, 37.5% of the fast-twitch EDL fibers developed maximum activated tensions (77.1 +/- 10.4 kN/m2), which were significantly lower than those developed by controls (244.0 +/- 14.3 kN/m2), and the slow-twitch SOL fibers displayed a significantly higher sensitivity to Ca2+ (and Sr2+) than the controls. All fibers from diabetic rats, including the low-tension EDL fibers and higher Ca sensitivity SOL fibers displayed control-like electrophoretic profiles of the major myofibrillar proteins. Taken together with data from earlier studies on the effects of long-term diabetes on whole skeletal muscle contractility, these results strongly suggest that 1) the decrease in tetanic tension output of EDL muscles induced by diabetes is caused mainly by direct effects of the diabetic condition on the contractile/regulatory system of a subpopulation of fast-twitch fibers, which develop little force, and 2) the diabetes-induced slowing of twitch times of SOL muscles is caused in part by the increased sensitivity to Ca2+ of the contractile apparatus in the slow-twitch fibers.

Analysis of Variance↗
PubMed1993

Endogenous MLC2 phosphorylation and Ca(2+)-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 Ca(2+)-activated force. The results show that (1) about 50% of MLC2 were 32P-phosphorylated after activation of the skinned fibre preparation by 30 microM [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 microM; 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 Kd for MgATP of the endogenous MLC2 phosphorylating system was estimated to be less than 300 microM. 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.

Adenosine Triphosphate↗
PubMed1993

Muscle samples obtained by needle biopsy are suitable for studying single fibre contractile properties.

Recent speculation as to whether material obtained by needle biopsy can be used to study physiological properties of single muscle fibres, led us to investigate whether fibre segments obtained by this procedure, exhibit the same contractile characteristics as those fibre segments obtained from ablated whole muscles. Needle biopsies were taken of the upper hindlimb from adult, Sprague-Dawley rats, killed by diethyl ether overdose. Single muscle fibre segments were isolated by microdissection, mechanically skinned (sarcolemma removed) and attached to a sensitive force recording transducer to measure Ca(2+)- and Sr(2+)-activated responses. No significant differences were found in Ca(2+)- and Sr(2+)-activated contractile characteristics (including maximum force generation) between fibres sampled by needle biopsy and those dissected from ablated intact muscles. Needle biopsied muscle fibre segments maintained their characteristic properties for up to 6-8 h (after sampling) if kept at room temperature in paraffin oil, and for up to 36 h if refrigerated (2-5 degrees C) in paraffin oil immediately after the biopsy procedure. It is concluded that muscle fibres sampled by needle biopsy have similar properties to fibres isolated from ablated muscles.

Animals↗
PubMed1993

Ca2+ levels in myotubes grown from the skeletal muscle of dystrophic (mdx) and normal mice.

1. Myotubes were grown in culture from normal (C57BL/ScSn) and mdx mice and the cytosolic [Ca2+] was monitored through development (5-21 days in culture) using fura-2 loaded via ionophoresis. Simultaneous measurements of the membrane potential and cytosolic [Ca2+] were made in normal and mdx myotubes before, during and after stimulation by action potentials elicited following anode break excitation. All experiments were undertaken at 22 degrees C. All data are expressed as means +/- S.E.M. 2. A new method was developed which enabled accurate determination of the fluorescence characteristics of fura-2 in murine skeletal muscle fibres. In the under in vitro conditions by 14.60 +/- 0.05, 9.40 +/- 0.15 and 6.90 +/- 0.43% respectively. 3. The resting cytosolic [Ca2+] in the mdx myotubes was consistently higher than in the normal myotubes throughout the developmental period measured. Overall, the resting cytosolic [Ca2+] in mdx myotubes (134 +/- 9 nM, n = 22) was twofold higher than in normal myotubes (66 +/- 6 nM, n = 26). After stimulation (one to three action potentials) the cytosolic [Ca2+] of both mdx and normal myotubes remained elevated. The mdx myotubes (236 +/- 55 nM, n = 5) again had approximately double the cytosolic [Ca2+] of normal myotubes (109 +/- 19 nM, n = 9). 4. The time course and amplitude of the Ca2+ responses measured in the mdx and normal myotubes after action potential stimulation were variable. Two categories of Ca2+ response were observed in mdx and normal myotubes, the first consisted of a small, slow rise in [Ca2+] that remained elevated and the second consisted of a rapid (time to peak 7.4 +/- 1.5 ms) (n = 8) rise in [Ca2+] with amplitudes in the range 61-773 nM and a [Ca2+] decay rate constant of 4.35 +/- 1.57 s-1 (n = 8) (range 0.96-15 s-1). 5. In conclusion, the elevated cytosolic [Ca2+] reported here through development of cultured mdx myotubes suggests that this genetic disorder results in a defect which compromises the ability of the myotubes to strictly regulate cytosolic [Ca2+]. The results are consistent with the presence of functionally abnormal Ca2+ channels recently reported in mdx myotubes.

Animals↗
PubMed1993

Contractile properties of skinned muscle fibres from young and adult normal and dystrophic (mdx) mice.

1. Single muscle fibres were enzymatically isolated from the soleus and extensor digitorum longus (EDL) muscles of genetically dystrophic mdx and normal (C57BL/10) mice aged 3-6 or 17-23 weeks. 2. Fibres of both muscles were chemically skinned with the non-ionic detergent Triton X-100 (2% v/v). Ca(2+)- and Sr(2+)-activated contractile responses were recorded and comparisons were made between several contractile parameters of various fibre types of normal and dystrophic mice of similar age. 3. There were no significant differences in the following contractile parameters of skinned fibres of normal and mdx mice of the same age: sensitivity to activating Ca2+ (pCa50) or Sr2+ (pSr50) and differential sensitivity to the activating ions (pCa50-pSr50). However the maximum isometric tension (Po) and the frequency of myofibrillar force oscillations in EDL fast-twitch fibres of young mdx mice were significantly lower than those of soleus fast-twitch fibres of the same animals, or fast-twitch fibres (EDL or soleus) of normal mice. 4. Age-related differences were apparent in some contractile parameters of both normal and mdx mice. In particular the steepness of force-pCa and force-pSr curves increased with age in normal mice, yet decreased with age in fibres of mdx mice. 5. A fluorescent probe, ethidium bromide, which interchelates with DNA, was used with laser-scanning confocal microscopy to determine the distribution of myonuclei in fibres. Fibres isolated from either muscle type of normal animals displayed a characteristic peripheral spiral of myonuclei. Fibres from muscles of mdx mice displayed three major patterns of nuclear distribution; the normal peripheral spiral, long central strands of nuclei, and a mixture of these two patterns. 6. The contractile characteristics of mdx fibres were not markedly influenced by the nuclear distribution pattern in that there were no discernible differences in the major contractile parameters (the Hill coefficients nCa and nSr, which are associated with the steepness of the Ca2+ and Sr2+ activation curves, pCa50, pSr50, pCa50-pSr50) of skinned fibres possessing peripheral or central nuclei. However, except for nSr, these values were all lower in individual fibres which displayed similar proportions of central and peripheral nuclei. The presence of mixed nucleation and absence of fibres with embryonic contractile characteristics in mdx mice suggest that the dystrophin-negative fibres can repair locally occurring muscle damage.

Aging↗
PubMed1993

Ca2+ and Sr2+ activation properties of skinned muscle fibres with different regulatory systems from crustacea and rat.

1. The contractile activation properties of long- (sarcomere length (SL) > 6 microns) and short- (SL < 4 microns) sarcomere fibres from the claw muscle of the yabby (freshwater crustacean, Cherax destructor) and the fast- and slow-twitch fibres from the rat have been investigated using single skinned muscle fibres activated in solutions containing Ca2+ or Sr2+ or both Ca2+ and Sr2+. 2. Sr2+ could not fully activate the contractile apparatus of either the long- or the short-sarcomere yabby preparations and the force-pSr curves for both fibre types were biphasic in shape. 3. The long- and short-sarcomere fibres from the yabby differed in their Ca(2+)- and Sr(2+)-activation properties. Thus the long-sarcomere fibres required a significantly lower [Ca2+] to produce 10% maximum force, had Ca(2+)-activation curves which were significantly shallower, and had a significantly higher ratio between maximum Sr(2+)- and maximum Ca(2+)-activated force than the short-sarcomere fibres. 4. Simultaneous activation with Ca2+ and Sr2+ showed a synergistic effect in the rat muscle fibres where Sr2+ could fully replace Ca2+ in activation. In contrast the results with the long- and short-sarcomere fibres from the yabby showed that in some functional states Sr2+ binds but cannot (or can only partially) activate the regulatory system while in others Sr2+ can fully replace Ca2+ in activating contraction. 5. The synergistic effect between Sr2+ and Ca2+ in mammalian muscle could be quantitatively explained if all regulatory sites involved in contractile activation have a similar value for the ratio between their affinity for Ca2+ and Sr2+. 6. Three distinguishable functional states (based on the fibre's ability to be activated by Sr2+ and Ca2+) were identified in the long-sarcomere fibres from the yabby: one where both Ca2+ and Sr2+ were able to activate contraction and had a relatively high sensitivity to Sr2+; one where both Ca2+ and Sr2+ were able to activate contraction but where the sensitivity to Sr2+ was lower and was more sensitive to a decrease in ionic strength; and one where Sr2+ binding to the regulatory system could not activate contraction. Equivalent states of the three described for the long-sarcomere fibres were also found in the short-sarcomere preparations. However, the short-sarcomere fibres had, in addition, a fourth state which was characterized by the ability of Sr2+ to activate contraction at reduced ionic strength but not at standard ionic strength.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Effects of osmolality and ionic strength on the mechanism of Ca2+ release in skinned skeletal muscle fibres of the toad.

1. The effects of increased osmolality and ionic strength on the mechanism of Ca2+ release were examined in mechanically skinned skeletal muscle fibres of the toad at 23 degrees C. Ca2+ release was induced by depolarizing the transverse tubular (T-) system by ionic substitution. 2. Increasing the osmolality of the 'myoplasmic' solution about four times (to 955 mosmol/kg), by addition of 700 mM sucrose to the standard potassium (K-)HDTA solution (HDTA: hexamethylenediamine-tetraacetate), only depressed the depolarization-induced response by about 46%. Much of this decrease could be attributed to a reduction in the Ca(2+)-sensitivity of the contractile proteins at this high osmolality. 3. Addition of > 400 mM sucrose itself often induced substantial Ca2+ release and a transient tension response. This 'spontaneous' release was (a) greatly enhanced when the sarcoplasmic reticulum (SR) had been heavily loaded with Ca2+, (b) little affected by inactivation of the voltage sensors by prolonged or permanent depolarization of the T-system and (c) blocked by Ruthenium Red (10 microM). 4. When both the osmolality and ionic strength were increased, by increasing the K-HDTA concentration, the depolarization-induced force was greatly reduced (to 35% at 818 mosmol/kg and 5% at 1095 mosmol/kg). Most of this reduction could be directly attributed to the substantially reduced maximum force and Ca2+ sensitivity of the contractile apparatus. 5. The small amount of releasable Ca2+ remaining in the SR after a single depolarization in a high-HDTA solution with 1 mM EGTA (to chelate the released Ca2+), indicated that depolarization could still elicit massive Ca2+ release at high ionic strength and osmolality (at 1 mM free Mg2+). 6. In contrast, when the total Mg2+ and ATP concentrations were raised about threefold (free [Mg2+] increased 2.7-fold) along with the osmolality and ionic strength, the ability of depolarization to elicit Ca2+ release was greatly hindered. 7. Osmotic compression of the skinned fibres to their in situ diameter by addition of 4% polyvinylpyrrolidone (PVP-40), substantially potentiated the depolarization-induced force responses, due partly to an increase in the Ca(2+)-sensitivity of the contractile apparatus. 8. These results indicate how increased intracellular osmolality, ionic strength and [Mg2+] produce the transient contraction and subsequent inhibition of tetanic tension in intact muscle fibres exposed to hypertonic solutions.

Animals↗
PubMed1993

Effects of halothane, enflurane, and isoflurane on skinned rat myocardium activated by Ca2+.

The effects of halothane (1.9 mM, 9.4 mM), enflurane (3.3 mM, 16.5 mM), and isoflurane (1.6 mM, 8.1 mM) on maximal Ca(2+)-activated force and Ca2+ sensitivity were studied in rat myocardial preparations rendered permeable by various methods. In preparations permeabilized either by mild homogenization or by saponin (50 micrograms/ml, 30 min), further disruption of the sarcolemma with 2% Triton X-100 resulted in increased maximal force and Ca2+ sensitivity. When membranes were mechanically disrupted or saponized, each vapor agent caused the myocardium to be more sensitive to Ca2+. In these preparations, maximal force was decreased by halothane but was not affected by enflurane or isoflurane. With cellular membranes completely disrupted by Triton, the previously observed increase in Ca2+ sensitivity caused by enflurane was reduced, whereas that caused by halothane and isoflurane was abolished and maximal force was decreased by halothane and isoflurane but was not affected by enflurane. These results indicate that 1) components associated with the cellular membrane systems normally modulate force in mammalian myocardium, and 2) halothane, enflurane, and isoflurane have complex effects on these components. These results therefore can explain some of the differences in inotropic effects that these agents exert on mammalian myocardium.

Animals↗
PubMed1992

Abnormalities in structure and function of limb skeletal muscle fibres of dystrophic mdx mice.

In this study we have shown that the skeletal muscle fibres from adult (older than 26 weeks) mdx mice have gross structural deformities. We have characterized the onset and age dependence of this feature in mdx mice. The three dimensional structure of these deformities has been visualized in isolated fibres and the orientation of these deformities was determined within the muscle by confocal laser scanning microscopy. We have also shown that the occurrence of morphologically abnormal fibres is greater in muscles with longer fibres (extensor digitorum longus (EDL) and soleus, 6-7.3 mm long), than in muscles with shorter fibres (flexor digitorum brevis (FDB), 0.3-0.4 mm long). A population of post-degenerative fibres, with both central and peripheral nuclei coexistent along the length of the fibre, has also been identified in the muscles studied. We showed that a mild protocol of lengthening (eccentric) contractions (the muscle was stretched by 12% during a tetanic contraction) caused a major reduction in the maximal tetanic force subsequently produced by mdx EDL muscle. In contrast, maximal tetanic force production in normal soleus, normal EDL and mdx soleus muscles was not altered by this protocol. We suggest that the deformed fast glycolytic fibres which are found in adult mdx EDL but not in adult mdx soleus muscles are the population of fibres damaged by the lengthening protocol.

Aging↗
PubMed1992

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 of force 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1992

Differences in maximal activation properties of skinned short- and long-sarcomere muscle fibres from the claw of the freshwater crustacean Cherax destructor.

Single fibres of different sarcomere length at rest have been isolated from the claw muscle of the yabby (Cherax destructor), a decapod crustacean. Fibres of either long (SL > 6 microns) or short (SL < 4 microns) sarcomere length have been mechanically skinned and were maximally activated by Ca2+ and Sr2+ under various experimental conditions (ionic strength, in the presence of 2,3 butanedione monoxime (BDM)) to determine differences in their contractile properties. Isometric force was measured simultaneously with either myofibrillar MgATPase or fibre stiffness in both fibre types. The ultrastructure of individual long- and short-sarcomere fibres was also determined by electron microscopy. The long-sarcomere fibres developed greater tension (30.48 +/- 1.72 N cm-2) when maximally activated by Ca2+ compared with the short-sarcomere fibres (18.60 +/- 0.80 N cm-2). The difference in the maximum Ca(2+)-activated force can be explained by the difference in the amount of filament overlap between the two fibre types. The maximum Ca(2+)-activated myofibrillar MgATPase rate in the short-sarcomere fibres (1.60 +/- 0.27 mmol ATP l-1s-1) was higher, but not significantly different from the ATPase rate in fibres with long-sarcomeres (1.09 +/- 0.14 mmol ATP l-1s-1). As the concentration of myosin is estimated to be higher only by a factor of 1.22 in the short-sarcomere preparations there is no evidence to suggest that the myofibrillar MgATPase activity is different in the long- and short-sarcomere preparations. The maximum Ca(2+)-activated force (P0) of both short- and long-sarcomere fibres was quite insensitive to BDM compared with vertebrate muscle. Force decreased to 60.2 +/- 5.3% and 76.1 +/- 2.7% in the short- and long-sarcomere fibres respectively in the presence of 100 mmol l-1 BDM. The difference in the force depression between the long- and short-sarcomere fibres is statistically significant (p < 0.05). Fibre stiffness during maximum Ca(2+)-activation expressed as percentage maximum force per nm per half sarcomere was higher by a factor of 3.5 in short-sarcomere fibres than in long-sarcomere fibres suggesting that the compliance of the filaments in the long-sarcomere fibres is considerably higher than in the short-sarcomere fibres. Sr2+ could not activate the contractile apparatus to the same level as that seen by Ca2+ in either fibre type: the maximum Sr(2+)-activated force was (20 +/- 3%) and (63 +/- 3%) of the maximum Ca(2+)-activated force response in short- and long-sarcomere fibres, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Actins↗
PubMed1992

Effect of myoplasmic pH on excitation-contraction coupling in skeletal muscle fibres of the toad.

1. The effect of pH on excitation-contraction coupling in skeletal muscle of the toad was examined using a skinned fibre preparation which gives ready access to the intracellular environment while still allowing stimulation of Ca2+ release by the normal voltage-sensor mechanism. 2. In each fibre, depolarization-induced responses (produced by changing the ions in the bathing solution) were examined first at pH 7.1, and then at another pH between 6.1 and 8.0. At all pH levels examined, the first depolarization elicited a large response which was slightly greater (pH 7.6 and 8.0) or smaller (pH 6.6 and 6.1) than that at pH 7.1. The size of the first depolarization-induced response varied with pH in almost exactly the same manner as did the maximum Ca(2+)-activated response. The duration of the depolarization-induced response at all other pH levels was longer than at pH 7.1. 3. Repeated depolarizations (30 s or more apart) produced similar responses at pH 7.1, but at all other pH levels examined the second and subsequent responses became progressively smaller. The reasons for this decrease were different at low and high pH. 4. Examination of the size of the depolarization-induced response after reloading the depleted sarcoplasmic reticulum (SR) with Ca2+ in solutions at various pH levels, indicated that the SR Ca2+ pump operated more poorly at pH 6.6 and 6.1 than at pH 7.1. This can account for the wide and successively smaller depolarization-induced responses at acidic pH. 5. Examination of the size of the depolarization-induced response after 5 min bathing without stimulation in a very low [Ca2+] solution (-log10[Ca2+] (pCa) greater than 8.0, 1 mM-BAPTA (1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), indicated that the sarcoplasmic reticulum (SR) lost far more Ca2+ at pH 7.6 and 8.0 than at pH 7.1 or 6.1. This indicates that SR is 'leakier' at alkaline pH. 6. Exposure of fibres heavily loaded with Ca2+ to solutions at pH 8.0 invariably induced a large response within 1-6 s. This response was unaffected by inactivation of the voltage sensors and was blocked completely by 2 microM-Ruthenium Red. This response and the 'leak' at alkaline pH are consistent with previous studies of the pH dependence of Ca(2+)-activated opening of single Ca2+ release channels in lipid bilayers. 7. These findings indicate that depolarization can induce massive Ca2+ release at both acidic and alkaline pH, provided that the SR is normally loaded with Ca2+, and give further insight into the intracellular events underlying fatigue in skeletal muscle.

Animals↗
PubMed1991

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 Ca(2+)- and Sr(2+)-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.

Animals↗
PubMed1991

Effect of Mg2+ on the control of Ca2+ release in skeletal muscle fibres of the toad.

1. The effect of myoplasmic Mg2+ on Ca2+ release was examined in mechanically skinned skeletal muscle fibres, in which the normal voltage-sensor control of Ca2+ release is preserved. The voltage sensors could be activated by depolarizing the transverse tubular (T-) system by lowering the [K+] in the bathing solution. 2. Fibres spontaneously contracted when the free [Mg2+] was decreased from 1 to 0.05 mM, with no depolarization or change of total ATP, [Ca2+] or pH (pCa 6.7, 50 microM-EGTA). After such a 'low-Mg2+ response' the sarcoplasmic reticulum (SR) was depleted of Ca2+ and neither depolarization nor caffeine (2 mM) could induce a response, unless the [Mg2+] was raised and the SR reloaded with Ca2+. Exposure to 0.05 mM-Mg2+ at low [Ca2+] (2 mM-free EGTA, pCa greater than 8.7) also induced Ca2+ release and depleted the SR. 3. The response to low [Mg2+] was unaffected by inactivation of the voltage sensors, but was completely blocked by 2 microM-Ruthenium Red indicating that it involved Ca2+ efflux through the normal Ca2+ release channels. 4. In the absence of ATP (and creatine phosphate), complete removal of Mg2+ (i.e. no added Mg2+ with 1 mM-EDTA) did not induce Ca2+ release. Depolarization in the absence of Mg2+ and ATP also did not induce Ca2+ release. 5. Depolarization in 10 mM-Mg2+ (pCa 6.7, 50 microM-EGTA, 8 mM-total ATP) did not produce any response. In the presence of 1 mM-EGTA to chelate most of the released Ca2+, depolarizations in 10 mM-Mg2+ did not noticeably deplete the SR of Ca2+, whereas a single depolarization in 1 mM-Mg2+ (and 1 mM-EGTA) resulted in marked depletion. Depolarization in the presence of D600 and 10 mM-Mg2+ produced use-dependent 'paralysis', indicating that depolarization in 10 mM-Mg2+ did indeed activate the voltage sensors. 6. Depolarization in the presence of 10 mM-Mg2+ and 25 microM-ryanodine neither interfered with the normal voltage control of Ca2+ release nor caused depletion of the Ca2+ in the SR even after returning to 1 mM-Mg2+ for 1 min, indicating that few if any of the release channels had been opened by the depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗