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  • 1
    ISSN: 1573-2657
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary 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 μm) or short (SL 〈 4 μm) 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 Ca2+-activated force can be explained by the difference in the amount of filament overlap between the two fibre types. The maximum Ca2+-activated myofibrillar MgATPase rate in the short-sarcomere fibres (1.60±0.27 mmol ATP l−1 s−1) was higher, but not significantly different from the ATPase rate in fibres with long-sarcomeres (1.09±0.14 mmol ATP l−1 s−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 Ca2+-activated force (P 0) 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 Ca2+-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 Sr2+-activated force was (20±3%) and (63±3%) of the maximum Ca2+-activated force response in short- and long-sarcomere fibres, respectively. The ratio between fibre stiffness in the maximum Sr2+-activating solution and the Ca2+-activating solution was very similar to the ratio between the maximum Sr2+-activated force and Ca2+-activated force in either type of fibres, suggesting that the number of attached crossbridges is lower in the fibres when maximally activated by Sr2+ than when maximally activated by Ca2+. The short-sarcomere fibres were also more sensitive to changes in ionic strength than long-sarcomere fibres. In conclusion these results indicate that while several important specific characteristics of the short- and long-sarcomere length fibres (ATPase, maximum Ca2+-activated force and fibre stiffness) can be explained solely on differences in the ultrastructure (length and density per cross-sectional area of myosin filaments) there are also differences in the properties of the proteins involved in the force production and regulation evidenced by the differential effect of Sr2+, BDM and ionic strength on contractile activation in the two fibre types.
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  • 2
    ISSN: 1573-2657
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary In this study, we investigated the effect of the Ca2+ pump inhibitor, 2,5-di-(tert-butyl)-1,4-hydroquinone on the function of the contractile apparatus, Ca2+ uptake, the permeability of the sarcoplasmic reticulum to Ca2+ and excitation-contraction coupling, in mechanically skinned muscle fibres of the rat and toad. 2,5-di-(tert-butyl)-1,4-hydroquinone had no significant effect on the maximum force and Ca2+ sensitivity of the contractile apparatus in rat and toad fibres at concentrations of 20 and 5 μM respectively. In rat fibres, 2,5-di-(tert-butyl)-1,4-hydroquinone was found to inhibit sarcoplasmic reticulum Ca2+ loading in a dose dependent manner, with a half maximal effect at 2 μM. In toad fibres, 5 μM 2,5-di-(tert-butyl)-1,4-hydroquinone completely blocked sarcoplasmic reticulum Ca2+ loading. Exposure to 5 mM BAPTA revealed a small resting sarcoplasmic reticulum Ca2+ leak in unstimulated rat fibres. This Ca2+ leak was not significantly affected by the presence of 20 μM 2,5-di-(tert-butyl)-1,4-hydroquinone, suggesting that 2,5-di-(tert-butyl)-1,4-hydroquinone does not substantially block or activate the sarcoplasmic reticulum Ca2+ release channels. Depolarisation-induced force responses elicited in rat and toad skinned fibres were not significantly affected by 0.5 μM 2,5-di-(tert-butyl)-1,4-hydroquinone. In the rat fibres, 5 and 20 μM 2,5-di-(tert-butyl)-1,4-hydroquinone greatly increased the peak and duration of initial depolarisation-induced force responses, while subsequent responses were reduced. 2,5-di-(tert-butyl)-1,4-hydroquinone did not affect excitation contraction coupling, as depolarisation-induced force responses similar to initial controls could be elicited after 2,5-di-(tert-butyl)-1,4-hydroquinone exposure, provided that the initial Ca2+ release in 2,5-di-(tert-butyl)-1,4-hydroquinone was chelated with 0.5 mM EGTA (to prevent Ca2+-dependent damage) and the sarcoplasmic reticulum was reloaded with Ca2+. In the toad fibres, 5 μM 2,5-di-(tert-butyl)-1,4-hydroquinone had a similar effect on depolarisation-induced force responses to that observed at 20 μM 2,5-di-(tert-butyl)-1,4-hydroquinone in rat fibres. This study shows that 2,5-di-(tert-butyl)-1,4-hydroquinone specifically and reversibly inhibits the sarcoplasmic reticulum Ca2+ pump in skeletal muscle and therefore, 2,5-di-(tert-butyl)-1,4-hydroquinone could be a valuable tool for investigating the role of the sarcoplasmic reticulum in Ca2+ homeostasis in skeletal muscle.
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  • 3
    ISSN: 1573-2657
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: 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.
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  • 4
    ISSN: 1573-2657
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary To understand how the coexistence of fast and slow contractile and regulatory systems within single skeletal muscle fibres might affect contractile behaviour, fibre segments from the fast-twitch extensor digitorum longus and predominantly slow-twitch soleus muscle of the adult rat were tied together, either in parallel or in series, and then activated in Ca2+-and Sr2+-buffered solutions. Experimental force-pCa and force-pSr relations were compared with theoretical force-pCa and force_pSr curves predicted by a model for composite fibres, which accounted for the coexistence of fast and slow myosin within the contractile unit and enabled an estimate to be made of the relative contribution of fast- and slow-twitch elements within the tied-fibre combinations. The contractile behaviour of a fast-twitch and a slow-twitch muscle fibre tied either in series or in parallel, were compared with the force-pCa and force-pSr data predicted from the composite fibre model. Interestingly, the resultant force-pCa(-pSr) curves of the parallel-tied fibre combinations were well fitted with those predicted by the composite model. However, the experimental force-pCa(-pSr) curves of the series-tied fibres were not well fitted by a composite curve based on the known proportion of fast- and slow-twitch fibre components. A total force-length diagram was devised to take into account changes in the length of the fibre segments tied in series during activation, as well as possible differences in fibre diameter. Using this diagram it was possible to explain accurately the Ca2+ and Sr2+ activation curves of known fast- and slow-twitch segments tied in series. The results from this study are important for the interpretation of contractile date obtained from single muscle fibres exhibiting mixed fast- and slow-twitch contractile characteristics. Such muscle fibres have previously been identified in animals affected by muscular diseases (e.g. dystrophy), in mammalian extraocular muscles and in animals subjected to long-term exercise training.
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  • 5
    ISSN: 1573-2657
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The rate, magnitude and pharmacology of inorganic phosphate (Pi) transport into the sarcoplasmic reticulum were estimated in single, mechanically skinned skeletal muscle fibres of the rat. This was done, indirectly, by using a technique that measured the total Ca2+ content of the sarcoplasmic reticulum and by taking advantage of the 1:1 stoichiometry of Ca2+ and Pi transport into the sarcoplasmic reticulum lumen during Ca--Pi precipitation- induced Ca2+ loading. The apparent rate of Pi entry into the sarcoplasmic reticulum increased with increasing myoplasmic [Pi] in the 10 mm--50 mm range at a fixed, resting myoplasmic pCa of 7.15, as judged by the increase in the rate of Ca--Pi precipitation-induced sarcoplasmic reticulum Ca2+ uptake. At 20 mm myoplasmic [Pi] the rate of Pi entry was calculated to be at least 51 μm s−1 while the amount of Pi loaded appeared to saturate at around 3.5 mm (per fibre volume). These values are approximations due to the complex kinetics of formation of different species of Ca--Pi precipitate formed under physiological conditions. Phenylphosphonic acid (PhPA, 2.5 mm inhibited Pi transport by 37% at myoplasmic pCa 6.5 and also had a small, direct inhibitory effect on the sarcoplasmic reticulum Ca2+ pump (16%). In contrast, phosphonoformic acid (PFA, 1 mm) appeared to enhance both the degree of Pi entry and the activity of the sarcoplasmic reticulum Ca2+ pump, results that were attributed to transport of PFA into the sarcoplasmic reticulum lumen and its subsequent complexation with Ca2+. Thus, results from these studies indicate the presence of a Pi transporter in the sarcoplasmic reticulum membrane of mammalian skeletal muscle fibres that is (1) active at physiological concentrations of myoplasmic Pi and Ca2+ and (2) partially inhibited by PhPA. This Pi transporter represents a link between changes in myoplasmic [Pi] and subsequent changes in sarcoplasmic reticulum luminal [Pi]. It might therefore play a role in the delayed metabolic impairment of sarcoplasmic reticulum Ca2+ release seen during muscle fatigue, which should occur abruptly once the Ca--Pi solubility product is exceeded in the sarcoplasmic reticulum lumen
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  • 6
    ISSN: 1573-2657
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The main objective of this study was to analyse glycogen in single muscle fibres, using a recently developed microfluorometric method which detects subpicomol amounts of NADPH, glucose and glycogen (as glucosyl units) (detection limit 0.16–0.17pmol in a 25nl sample) without fluorochrome amplification. The fibres were freshly dissected from the twitch region of the iliofibularis muscle of the cane toad (Bufo marinus), and were mechanically skinned under paraffin oil to gain access to the intracellular compartments. The results show that: (1) glycogen concentrations in toad skeletal muscle fibres range between 25.8 and 369mmol glucosyl units/litre fibre volume; (2) there is a large variation in glycogen content between individual fibres from the iliofibularis muscle of one animal; (3) there are seasonal differences in the glycogen content of toad single muscle fibres; (4) the total amount of glycogen in single muscle fibres of the toad does not decrease significantly when storing the tissue, under paraffin oil, at 20–25°C for up to 6h or at 4°C for up to 24h; and (5) 15–26% of fibre glycogen can be washed in an aqueous solution at pH 5–7, within 5min, while 74–85% of fibre glycogen remains associated with the washed skinned fibre, even after 40min exposure of the skinned fibre preparation to the aqueous environment. The retention of most glycogen in the fibre preparation after mechanical removal of the plasma membrane and extensive washing indicates that in toad skeletal muscle fibres the largest proportion of glycogen is tightly bound to intracellular structures. The results also show that the skinned muscle fibre preparation is well suited for microfluorometric glycogen determination, since low molecular weight non-glycogen contributors to the fluorescence signal can be removed from the myoplasmic space prior to the glycogen hydrolysis step.
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  • 7
    Publication Date: 2014-01-01
    Print ISSN: 0006-3495
    Electronic ISSN: 1542-0086
    Topics: Biology , Physics
    Published by Elsevier
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  • 8
    Publication Date: 2009-07-01
    Print ISSN: 0006-291X
    Electronic ISSN: 1090-2104
    Topics: Biology , Chemistry and Pharmacology , Physics
    Published by Elsevier
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  • 9
    Publication Date: 2009-02-01
    Print ISSN: 0006-3495
    Electronic ISSN: 1542-0086
    Topics: Biology , Physics
    Published by Elsevier
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