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D George Stephenson

Publications and source records attributed to D George Stephenson.

16 recordsLinked to original sources
PubMed2006

Mitochondrial superoxide production in skeletal muscle fibers of the rat and decreased fiber excitability.

Mammalian skeletal muscles generate marked amounts of superoxide (O(2)(.-)) at 37 degrees C, but it is not well understood which is the main source of O(2)(.-) production in the muscle fibers and how this interferes with muscle function. To answer these questions, O(2)(.-) production and twitch force responses were measured at 37 degrees C in mechanically skinned muscle fibers of rat extensor digitorum longus (EDL) muscle. In mechanically skinned fibers, the sarcolemma is removed avoiding potential sources of O(2)(.-) production that are not intrinsically part of the muscle fibers, such as nerve terminals, blood cells, capillaries and other blood vessels in the whole muscle. O(2)(.-) production was also measured in split single EDL muscle fibers, where part of the sarcolemma remained attached, and small bundles of intact isolated EDL muscle fibers at rest, in the presence and absence of modifiers of mitochondrial function. The results lead to the conclusion that mitochondrial production of O(2)(.-) accounts for most of the O(2)(.-) measured intracellularly or extracellularly in skeletal muscle fibers at rest and at 37 degrees C. Muscle fiber excitability at 37 degrees C was greatly improved in the presence of a membrane permeant O(2)(.-) dismutase mimetic (Tempol), demonstrating a direct link between O(2)(.-) production in the mitochondria and muscle fiber performance. This implicates mitochondrial O(2)(.-) production in the down-regulation of skeletal muscle function, thus providing a feedback pathway for communication between mitochondria and plasma membranes that is not directly related to the main function of mitochondria as the power plant of the mammalian muscle cell.

Animals↗
PubMed2006

Tubular system excitability: an essential component of excitation-contraction coupling in fast-twitch fibres of vertebrate skeletal muscle.

The tubular (t-) system is the main interface between the myoplasm and the extracellular environment and is responsible for the rapid inward spread of excitation from the sarcolemma to the inner parts of the skeletal muscle fibre as well as for signal transfer to the sarcoplasmic reticulum to release Ca2+ that, in turn, activates the contractile apparatus. In this review, I explore the insights provided by the mechanically skinned muscle fibre preparation to the better understanding of the importance of the t-system excitability in determining the force response under physiologically relevant conditions. In the mechanically skinned muscle fibre, the t-system seals off after is physically separated from the sarcolemma and its excitability can be investigated by electrical stimulation under controlled conditions. Parameters that can be assessed include the threshold for action potential generation, specific electrical resistance and time constant of the tubular wall, quantity of charge transferred during an action potential, refractory period, length constant and velocity of excitation propagation. Results obtained with mechanically skinned fibres from fast-twitch muscles show that decreased t-system excitability does not necessarily translate into reduced force output, but for any particular set of physiologically relevant conditions there is a level below which a further decrease in t-system excitability markedly decreases the force output. There are several built-in mechanisms linked to the metabolic/energetic state of the muscle fibre which prevent complete action potential failure in the t-system, thus allowing the muscle to respond to nerve stimulation, even if the response becomes markedly attenuated.

Action Potentials↗
PubMed2006

Myosin heavy chain isoform composition and stretch activation kinetics in single fibres of Xenopus laevis iliofibularis muscle.

Skeletal muscle is composed of specialized fibre types that enable it to fulfil complex and variable functional needs. Muscle fibres of Xenopus laevis, a frog formerly classified as a toad, were the first to be typed based on a combination of physiological, morphological, histochemical and biochemical characteristics. Currently the most widely accepted criterion for muscle fibre typing is the myosin heavy chain (MHC) isoform composition because it is assumed that variations of this protein are the most important contributors to functional diversity. Yet this criterion has not been used for classification of Xenopus fibres due to the lack of an effective protocol for MHC isoform analysis. In the present study we aimed to resolve and visualize electrophoretically the MHC isoforms expressed in the iliofibularis muscle of Xenopus laevis, to define their functional identity and to classify the fibres based on their MHC isoform composition. Using a SDS-PAGE protocol that proved successful with mammalian muscle MHC isoforms, we were able to detect five MHC isoforms in Xenopus iliofibularis muscle. The kinetics of stretch-induced force transients (stretch activation) produced by a fibre was strongly correlated with its MHC isoform content indicating that the five MHC isoforms confer different kinetics characteristics. Hybrid fibre types containing two MHC isoforms exhibited stretch activation kinetics parameters that were intermediate between those of the corresponding pure fibre types. These results clearly show that the MHC isoforms expressed in Xenopus muscle are functionally different thereby validating the idea that MHC isoform composition is the most reliable criterion for vertebrate skeletal muscle fibre type classification. Thus, our results lay the foundation for the unequivocal classification of the muscle fibres in the Xenopus iliofibularis muscle and for gaining further insights into skeletal muscle fibre diversity.

Animals↗
PubMed2005

Disruption of excitation-contraction coupling and titin by endogenous Ca2+-activated proteases in toad muscle fibres.

This study investigated the effects of elevated, physiological levels of intracellular free [Ca(2+)] on depolarization-induced force responses, and on passive and active force production by the contractile apparatus in mechanically skinned fibres of toad iliofibularis muscle. Excitation-contraction (EC) coupling was retained after skinning and force responses could be elicited by depolarization of the transverse-tubular (T-) system. Raising the cytoplasmic [Ca(2+)] to approximately 1 microm or above for 3 min caused an irreversible reduction in the depolarization-induced force response by interrupting the coupling between the voltage sensors in the T-system and the Ca(2+) release channels in the sarcoplasmic reticulum. This uncoupling showed a steep [Ca(2+)] dependency, with 50% uncoupling at approximately 1.9 microm Ca(2+). The uncoupling occurring with 2 microm Ca(2+) was largely prevented by the calpain inhibitor leupeptin (1 mm). Raising the cytoplasmic [Ca(2+)] above 1 microm also caused an irreversible decline in passive force production in stretched skinned fibres in a manner graded by [Ca(2+)], though at a much slower relative rate than loss of coupling. The progressive loss of passive force could be rapidly stopped by lowering [Ca(2+)] to 10 nm, and was almost completely inhibited by 1 mm leupeptin but not by 10 microm calpastatin. Muscle homogenates preactivated by Ca(2+) exposure also evidently contained a diffusible factor that caused damage to passive force production in a Ca(2+)-dependent manner. Western blotting showed that: (a) calpain-3 was present in the skinned fibres and was activated by the Ca(2+)exposure, and (b) the Ca(2+) exposure in stretched skinned fibres resulted in proteolysis of titin. We conclude that the disruption of EC coupling occurring at elevated levels of [Ca(2+)] is likely to be caused at least in part by Ca(2+)-activated proteases, most likely by calpain-3, though a role of calpain-1 is not excluded.

Animals↗
PubMed2005

Differential effects of arginine, glutamate and phosphoarginine on Ca(2+)-activation properties of muscle fibres from crayfish and rat.

The effects of two amino acids, arginine which has a positively charged side-chain and glutamate which has a negatively charged side-chain on the Ca2+-activation properties of the contractile apparatus were examined in four structurally and functionally different types of skeletal muscle; long- and short-sarcomere fibres from the claw muscle of the yabby (a freshwater decapod crustacean), and fast- and slow-twitch fibres from limb muscles of the rat. Single skinned fibres were activated in carefully balanced solutions of different pCa (-log10[Ca2+]) that either contained the test solute ("test") or not ("control"). The effect of phosphoarginine, a phosphagen that bears a nett negative charge, was also compared to the effects of arginine. Results show that (i) arginine (33-36 mmol l(-1)) significantly shifted the force-pCa curve by 0.08-0.13 pCa units in the direction of increased sensitivity to Ca2+-activated contraction in all fibre types; (ii) phosphoarginine (9-10 mmol l(-1)) induced a significant shift of the force-pCa curve by 0.18-0.24 pCa units in the direction of increased sensitivity to Ca2+ in mammalian fast- and slow-twitch fibres, but had no significant effects on the force-pCa relation in either long- or short-sarcomere crustacean fibres; (iii) glutamate (36-40 mmol l(-1)), like arginine affected the force-pCa relation of all fibre types investigated, but in the opposite direction, causing a significant decrease in the sensitivity to Ca2+-activated contraction by 0.08-0.19 pCa units; (iv) arginine, phosphoarginine and glutamate had little or no effect on the maximum Ca2+-activated force of crustacean and mammalian fibres. The results suggest that the opposing effects of glutamate and arginine are not related to simply their charge structure, but must involve complex interactions between these molecules, Ca2+ and the regulatory and other myofibrillar proteins.

Animals↗
PubMed2004

Intracellular acidosis enhances the excitability of working muscle.

Intracellular acidification of skeletal muscles is commonly thought to contribute to muscle fatigue. However, intracellular acidosis also acts to preserve muscle excitability when muscles become depolarized, which occurs with working muscles. Here, we show that this process may be mediated by decreased chloride permeability, which enables action potentials to still be propagated along the internal network of tubules in a muscle fiber (the T system) despite muscle depolarization. These results implicate chloride ion channels in muscle function and emphasize that intracellular acidosis of muscle has protective effects during muscle fatigue.

Action Potentials↗
PubMed2004

Effect of creatine on contractile force and sensitivity in mechanically skinned single fibers from rat skeletal muscle.

Increasing the intramuscular stores of total creatine [TCr = creatine (Cr) + creatine phosphate (CrP)] can result in improved muscle performance during certain types of exercise in humans. Initial uptake of Cr is accompanied by an increase in cellular water to maintain osmotic balance, resulting in a decrease in myoplasmic ionic strength. Mechanically skinned single fibers from rat soleus (SOL) and extensor digitorum longus (EDL) muscles were used to examine the direct effects on the contractile apparatus of increasing [Cr], increasing [Cr] plus decreasing ionic strength, and increasing [Cr] and [CrP] with no change in ionic strength. Increasing [Cr] from 19 to 32 mM, accompanied by appropriate increases in water to maintain osmolality, had appreciable beneficial effects on contractile apparatus performance. Compared with control conditions, both SOL and EDL fibers showed increases in Ca(2+) sensitivity (+0.061 +/- 0.004 and +0.049 +/- 0.009 pCa units, respectively) and maximum Ca(2+)-activated force (to 104 +/- 1 and 105 +/- 1%, respectively). In contrast, increasing [Cr] alone had a small inhibitory effect. When both [Cr] and [CrP] were increased, there was virtually no change in Ca(2+) sensitivity of the contractile apparatus, and maximum Ca(2+)-activated force was approximately 106 +/- 1% compared with control conditions in both SOL and EDL fibers. These results suggest that the initial improvement in performance observed with Cr supplementation is likely due in large part to direct effects of the accompanying decrease in myoplasmic ionic strength on the properties of the contractile apparatus.

Animals↗
PubMed2004

Troponin C isoform composition determines differences in Sr(2+)-activation characteristics between rat diaphragm fibers.

Single fibers of rat diaphragm containing different naturally occurring combinations of myofibrillar protein isoforms were used to evaluate the contribution of troponin C (TnC) isoforms to fiber type-related differences with respect to sensitivity to Sr(2+) of the contractile system. Mechanically skinned fibers were studied for their isometric force vs. Sr(2+) concentration ([Sr(2+)]) relationships and then analyzed electrophoretically for myofibrillar protein isoform composition. Our data demonstrate that fiber-type differences in Sr(2+) dependence of contractile activation processes are primarily determined by the TnC isoform composition, with the slow isoform conferring on average a sevenfold greater sensitivity to Sr(2+) than the fast isoform. Moreover, the ratio of TnC isoforms determined functionally from the force-pSr (-log(10) [Sr(2+)]) curves is tightly (r(2) = 0.97) positively correlated with that estimated electrophoretically. Together, these results validate the use of Sr(2+) activation characteristics to distinguish fibers containing different proportions of fast and slow TnC isoforms and to study the mechanisms by which divalent cations activate the contractile apparatus. We also found that the functionally and electrophoretically determined ratios of TnC isoforms present in a fiber display similar sigmoidal relationships with the ratio of myosin heavy chain (MHC) isoform types expressed. These relationships 1) offer further insight in the functional and molecular expression of TnC in relation to the molecular expression of MHC isoform types and 2) may provide the basis for predicting sensitivity to Sr(2+), TnC, and MHC isoforms in pure and hybrid skeletal muscle fibers.

Animals↗
PubMed2004

Osmotic properties of the sealed tubular system of toad and rat skeletal muscle.

A method was developed that allows conversion of changes in maximum Ca(2+)-dependent fluorescence of a fixed amount of fluo-3 into volume changes of the fluo-3-containing solution. This method was then applied to investigate by confocal microscopy the osmotic properties of the sealed tubular (t-) system of toad and rat mechanically skinned fibers in which a certain amount of fluo-3 was trapped. When the osmolality of the myoplasmic environment was altered by simple dilution or addition of sucrose within the range 190-638 mosmol kg(-1), the sealed t-system of toad fibers behaved almost like an ideal osmometer, changing its volume inverse proportionally to osmolality. However, increasing the osmolality above 638 to 2,550 mosmol kg(-1) caused hardly any change in t-system volume. In myoplasmic solutions made hypotonic to 128 mosmol kg(-1), a loss of Ca(2+) from the sealed t-system of toad fibers occurred, presumably through either stretch-activated cationic channels or store-operated Ca(2+) channels. In contrast to the behavior of the t-system in toad fibers, the volume of the sealed t-system of rat fibers changed little (by <20%) when the osmolality of the myoplasmic environment changed between 210 and 2,800 mosmol kg(-1). Results were also validated with calcein. Clear differences between rat and toad fibers were also found with respect to the t-system permeability for glycerol. Thus, glycerol equilibrated across the rat t-system within seconds to minutes, but was not equilibrated across the t-system of toad fibers even after 20 min. These results have broad implications for understanding osmotic properties of the t-system and reversible vacuolation in muscle fibers. Furthermore, we observed for the first time in mammalian fibers an orderly lateral shift of the t-system networks whereby t-tubule networks to the left of the Z-line crossover to become t-tubule networks to the right of the Z-line in the adjacent sarcomere (and vice versa). This orderly rearrangement can provide a pathway for longitudinal continuity of the t-system along the fiber axis.

Aniline Compounds↗
PubMed2003

A novel signalling pathway originating in mitochondria modulates rat skeletal muscle membrane excitability.

Single skeletal muscle fibres from rat and cane toad were mechanically skinned and stimulated either electrically by initiating action potentials in the sealed transverse (t-) tubular system or by ion substitution causing depolarisation of the t-system to pre-determined levels. Depression of mitochondrial ATP-producing function with three diverse mitochondrial function antagonists (azide: 1-10 mM; oligomycin 1 microg ml-1 and carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP) 1 microM), under conditions in which the cytosolic ATP was maintained high and constant, invariably reduced the excitability of rat fibres but had no obvious effect on the excitability of toad fibres, where mitochondria are less abundant and differently located. The reduction in excitability linked to mitochondria in rat fibres appears to be caused by depolarisation of the sealed t-system membrane. These observations suggest that mitochondria can regulate the functional state of mammalian muscle cells and have important implications for understanding how the balance between ATP utilisation and ATP production is regulated at the cellular level in general and in mammalian skeletal muscle fibres in particular.

Action Potentials↗
PubMed2003

Identification of the coupling between skeletal muscle store-operated Ca2+ entry and the inositol trisphosphate receptor.

Examination of store-operated Ca(2+) entry (SOC) in single, mechanically skinned skeletal muscle cells by confocal microscopy shows that the inositol 1,4,5-trisphosphate (IP(3)) receptor acts as a sarcoplasmic reticulum [Ca(2+)] sensor and mediates SOC by physical coupling without playing a key role in Ca(2+) release from internal stores, as is the case with various cell types in which SOC was investigated previously. The results have broad implications for understanding the mechanism of SOC that is essential for cell function in general and muscle function in particular. Moreover, the study ascribes an important role to the IP(3) receptors in skeletal muscle, the role of which with respect to Ca(2+) homeostasis was ill defined until now.

Animals↗
PubMed2003

Relationship between isometric force and myofibrillar MgATPase at short sarcomere length in skeletal and cardiac muscle and its relevance to the concept of activation heat.

1. This paper has been written in recognition of the seminal contributions to cardiac and skeletal muscle energetics made by Professor Colin Gibbs during his distinguished academic career. 2. The paper focuses on what is now known about the relationship between Ca2+-activated isometric force production and myofibrillar MgATPase in intact and skinned (surface membrane rendered permeable) skeletal and cardiac muscle preparations at short sarcomere lengths. 3. The relevance of this relationship to understanding the interactions between the actin and myosin filaments at the cross-bridge level in the region of double actin filament overlap and the cellular basis of 'activation heat' measurements in intact striated muscles is discussed.

Animals↗
PubMed2002

Reversible changes in Ca(2+)-activation properties of rat skeletal muscle exposed to elevated physiological temperatures.

Exposure of relaxed rat extensor digitorum longus (EDL; predominantly fast-twitch) muscle to temperatures in the upper physiological range for mammalian skeletal muscle (43-46 degrees C) led to reversible alterations of the contractile activation properties. These properties were studied using the mechanically skinned fibre preparation activated in Ca(2+)-buffered solutions. The maximum Ca(2+)-activated force (maximum force per cross-sectional area) and the steepness of force-pCa (-log(10)[Ca(2+)]) curves as measured by the Hill coefficient (n(H)) reversibly decreased by factors of 8 and 2.5, respectively, when the EDL muscle was treated at 43 degrees C for 30 min and 5 and 2.8, respectively, with treatment at 46 degrees C for 5 min. Treatment at 47 degrees C for 5 min produced an even more marked depression in maximum specific force, which fully recovered after treatment, and in the Hill coefficient, which did not recover after treatment. After all temperature treatments there was no change in the level of [Ca(2+)] at which 50 % maximum force was generated. The temperature-induced depression in force production and steepness of the force-pCa curves were shown to be associated with superoxide (O(2)(-)) production in muscle (apparent rate of O(2)(-) production at room temperature, 0.055 +/- 0.008 nmol min(-1) (g wet weight)(-1); and following treatment to 46 degrees C for 5 min, 1.8 +/- 0.2 nmol min(-1) (g wet weight)(-1)) because 20 mM Tiron, a membrane-permeant O(2)(-) scavenger, was able to markedly suppress the net rate of O(2)(-) production and prevent any temperature-induced depression of contractile parameters. The temperature-induced depression in force production of the contractile apparatus could be reversed either by allowing the intact muscle to recover for 3-4 h at room temperature or by treatment of the skinned fibre preparation with dithiothreitol (a potent reducing agent) in the relaxing solution. These results demonstrate that mammalian skeletal muscle has the ability to uncouple force production reversibly from the activator Ca(2+) as the temperature increases in the upper physiological range through an increase in O(2)(-) production.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗
PubMed2002

Tubular system volume changes in twitch fibres from toad and rat skeletal muscle assessed by confocal microscopy.

The volume of the extracellular compartment (tubular system) within intact muscle fibres from cane toad and rat was measured under various conditions using confocal microscopy. Under physiological conditions at rest, the fractional volume of the tubular system (t-sys(Vol)) was 1.38 +/- 0.09 % (n = 17), 1.41 +/- 0.09 % (n = 12) and 0.83 +/- 0.07 % (n = 12) of the total fibre volume in the twitch fibres from toad iliofibularis muscle, rat extensor digitorum longus muscle and rat soleus muscle, respectively. In toad muscle fibres, the t-sys(Vol) decreased by 30 % when the tubular system was fully depolarized and decreased by 15 % when membrane cholesterol was depleted from the tubular system with methyl-beta-cyclodextrin but did not change as the sarcomere length was changed from 1.93 to 3.30 microm. There was also an increase by 30 % and a decrease by 25 % in t-sys(Vol) when toad fibres were equilibrated in solutions that were 2.5-fold hypertonic and 50 % hypotonic, respectively. When the changes in total fibre volume were taken into consideration, the t-sys(Vol) expressed as a percentage of the isotonic fibre volume did actually decrease as tonicity increased, revealing that the tubular system in intact fibres cannot be compressed below 0.9 % of the isotonic fibre volume. The results can be explained in terms of forces acting at the level of the tubular wall. These observations have important physiological implications showing that the tubular system is a dynamic membrane structure capable of changing its volume in response to the membrane potential, cholesterol depletion and osmotic stress but not when the sarcomere length is changed in resting muscle.

Animals↗
PubMed2002

Properties of the vertebrate skeletal muscle tubular system as a sealed compartment.

Confocal imaging of impermeant fluorescent dyes trapped in the tubular (t-) system of skeletal muscle fibres of rat and cane toad was used to examine changes in the morphology of the t-system upon mechanical skinning, the time course of dye loss from the sealed t-system in mechanically skinned fibres and the influence of rapid application and removal of glycerol on the morphology of the sealed t-system. In contrast to intact fibres, which have a t-system open to the outside, the sealed t-system of toad mechanically skinned fibres consistently displayed local swellings (vesicles). The occurrence of vesicles in the sealed t-system of rat-skinned fibres was infrequent. Application and removal of 200-400 mM glycerol to the sealed t-system did not produce any obvious changes in its morphology. The dyes fluo-3, fura-2 and Oregon green 488 were lost from the sealed t-system of toad fibres at different rates suggesting that the mechanism of organic anion transport across the tubular wall was not by indiscriminate bulk transport. The rate of fluo-3 and fura-2 loss from the sealed t-system of rat fibres was greater in rat than in toad fibres and could be explained by differences in surface area: volume ratio of the t-system in the two fibre types. Based on the results presented here and on other results from this laboratory, an explanation is given for the formation of numerous vesicles in toad-skinned fibres and lack of vesicle formation in rat-skinned fibres. This explanation can also help with better understanding the mechanism responsible for vacuole formation in intact fibres.

Animals↗