Calcium and strontium contractile activation properties of single skinned skeletal muscle fibres from elderly women 66-90 years of age.

The single freshly skinned muscle fibre technique was used to investigate Ca2+- and Sr2+-activation properties of skeletal muscle fibres from elderly women (66-90 years). Muscle biopsies were obtained from the vastus lateralis muscle. Three populations of muscle fibres were identified according to their specific Sr2+-activation properties: slow-twitch (type I), fast-twitch (type II) and hybrid (type I/II) fibres. All three fibre types were sampled from the biopsies of 66 to 72 years old women, but the muscle biopsies of women older than 80 years yielded only slow-twitch (type I) fibres. The proportion of hybrid fibres in the vastus lateralis muscle of women of circa 70 years of age (24%) was several-fold greater than in the same muscle of adults (< 10%), suggesting that muscle remodelling occurs around this age. There were no differences between the Ca2+- and Sr2+-activation properties of slow-twitch fibres from the two groups of elderly women, but there were differences compared with muscle fibres from young adults with respect to sensitivity to Ca2+, steepness of the activation curves, and characteristics of the fibre-type dependent phenomenon of spontaneous oscillatory contractions (SPOC) (or force oscillations) occurring at submaximal levels of activation. The maximal Ca2+ activated specific force from all the fibres collected from the seven old women use in the present study was significantly lower by 20% than in the same muscle of adults. Taken together these results show there are qualitative and quantitative changes in the activation properties of the contractile apparatus of muscle fibres from the vastus lateralis muscle of women with advancing age, and that these changes need to be considered when explaining observed changes in women's mobility with aging.

Junctional membrane Ca2+ dynamics in human muscle fibers are altered by malignant hyperthermia causative RyR mutation.

We used the nanometer-wide tubules of the transverse tubular (t)-system of human skeletal muscle fibers as sensitive sensors for the quantitative monitoring of the Ca2+-handling properties in the narrow junctional cytoplasmic space sandwiched between the tubular membrane and the sarcoplasmic reticulum cisternae in single muscle fibers. The t-system sealed with a Ca2+-sensitive dye trapped in it is sensitive to changes in ryanodine receptor (RyR) Ca2+ leak, the store operated calcium entry flux, plasma membrane Ca pump, and sodium-calcium exchanger activities, thus making the sealed t-system a nanodomain Ca2+ sensor of Ca2+ dynamics in the junctional space. The sensor was used to assess the basal Ca2+-handling properties of human muscle fibers obtained by needle biopsy from control subjects and from people with a malignant hyperthermia (MH) causative RyR variant. Using this approach we show that the muscle fibers from MH-susceptible individuals display leakier RyRs and a greater capacity to extrude Ca2+ across the t-system membrane compared with fibers from controls. This study provides a quantitative way to assess the effect of RyR variants on junctional membrane Ca2+ handling under defined ionic conditions.

Measurement of force and calcium release using mechanically skinned fibers from mammalian skeletal muscle.

The mechanically skinned (or "peeled") skeletal muscle fiber technique is a highly versatile procedure that allows controlled examination of each of the steps in the excitation-contraction (EC)-coupling sequence in skeletal muscle fibers, starting with excitation/depolarization of the transverse tubular (T)-system through to Ca2+ release from sarcoplasmic reticulum (SR) and finally force development by the contractile apparatus. It can also show the overall response of the whole EC-coupling sequence together, such as in twitch and tetanic force responses. A major advantage over intact muscle fiber preparations is that it is possible to set and rapidly manipulate the "intracellular" conditions, allowing examination of the effects of key variables (e.g., intracellular pH, ATP levels, redox state, etc.) on each individual step in EC coupling. This Cores of Reproducibility in Physiology (CORP) article describes the rationale, procedures, and experimental details of the various ways in which the mechanically skinned fiber technique is used in our laboratory to examine the physiological mechanisms controlling Ca2+ release and contraction in skeletal muscle fibers and the aberrations and dysfunction occurring with exercise and disease.

NHE- and diffusion-dependent proton fluxes across the tubular system membranes of fast-twitch muscle fibers of the rat.

The complex membrane structure of the tubular system (t-system) in skeletal muscle fibers is open to the extracellular environment, which prevents measurements of H+ movement across its interface with the cytoplasm by conventional methods. Consequently, little is known about the t-system's role in the regulation of cytoplasmic pH, which is different from extracellular pH. Here we describe a novel approach to measure H+-flux measurements across the t-system of fast-twitch fibers under different conditions. The approach involves loading the t-system of intact rat fast-twitch fibers with a strong pH buffer (20 mM HEPES) and pH-sensitive fluorescent probe (10 mM HPTS) before the t-system is sealed off. The pH changes in the t-system are then tracked by confocal microscopy after rapid changes in cytoplasmic ionic conditions. T-system sealing is achieved by removing the sarcolemma by microdissection (mechanical skinning), which causes the tubules to pinch off and seal tight. After this procedure, the t-system repolarizes to physiological levels and can be electrically stimulated when placed in K+-based solutions of cytosolic-like ionic composition. Using this approach, we show that the t-system of fast-twitch skeletal fibers displays amiloride-sensitive Na+/H+ exchange (NHE), which decreases markedly at alkaline cytosolic pH and has properties similar to that in mammalian cardiac myocytes. We observed mean values for NHE density and proton permeability coefficient of 339 pmol/m2 of t-system membrane and 158 µm/s, respectively. We conclude that the cytosolic pH in intact resting muscle can be quantitatively explained with respect to extracellular pH by assuming that these values apply to the t-system membrane and the sarcolemma.

Changes in plasma membrane Ca-ATPase and stromal interacting molecule 1 expression levels for Ca(2+) signaling in dystrophic mdx mouse muscle.

The majority of the skeletal muscle plasma membrane is internalized as part of the tubular (t-) system, forming a standing junction with the sarcoplasmic reticulum (SR) membrane throughout the muscle fiber. This arrangement facilitates not only a rapid and large release of Ca(2+) from the SR for contraction upon excitation of the fiber, but has also direct implications for other interdependent cellular regulators of Ca(2+). The t-system plasma membrane Ca-ATPase (PMCA) and store-operated Ca(2+) entry (SOCE) can also be activated upon release of SR Ca(2+). In muscle, the SR Ca(2+) sensor responsible for rapidly activated SOCE appears to be the stromal interacting molecule 1L (STIM1L) isoform of STIM1 protein, which directly interacts with the Orai1 Ca(2+) channel in the t-system. The common isoform of STIM1 is STIM1S, and it has been shown that STIM1 together with Orai1 in a complex with the partner protein of STIM (POST) reduces the activity of the PMCA. We have previously shown that Orai1 and STIM1 are upregulated in dystrophic mdx mouse muscle, and here we show that STIM1L and PMCA are also upregulated in mdx muscle. Moreover, we show that the ratios of STIM1L to STIM1S in wild-type (WT) and mdx muscle are not different. We also show a greater store-dependent Ca(2+) influx in mdx compared with WT muscle for similar levels of SR Ca(2+) release while normal activation and deactivation properties were maintained. Interestingly, the fiber-averaged ability of WT and mdx muscle to extrude Ca(2+) via PMCA was found to be the same despite differences in PMCA densities. This suggests that there is a close relationship among PMCA, STIM1L, STIM1S, Orai1, and also POST expression in mdx muscle to maintain the same Ca(2+) extrusion properties as in the WT muscle.

Longitudinal and transversal propagation of excitation along the tubular system of rat fast-twitch muscle fibres studied by high speed confocal microscopy.

Mammalian skeletal muscle fibres possess a tubular (t-) system that consists of regularly spaced transverse elements which are also connected in the longitudinal direction. This tubular network provides a pathway for the propagation of action potentials (APs) both radially and longitudinally within the fibre, but little is known about the actual radial and longitudinal AP conduction velocities along the tubular network in mammalian skeletal muscle fibres. The aim of this study was to track AP propagation within the t-system network of fast-twitch rat muscle fibres with high spatio-temporal resolution when the t-system was isolated from the surface membrane. For this we used high speed confocal imaging of AP-induced Ca(2+) release in contraction-suppressed mechanically skinned fast-twitch fibres where the t-system can be electrically excited in the absence of the surface membrane. Supramaximal field pulses normally elicited a synchronous AP-induced release of Ca(2+) along one side of the fibre axis which propagated uniformly across the fibre. In some cases up to 80 or more adjacent transverse tubules failed to be excited by the field pulse, while adjacent areas responded with normal Ca(2+) release. In these cases a continuous front of Ca(2+) release with an angle to the scanning line was observed due to APs propagating longitudinally. From these observations the radial/transversal and longitudinal AP conduction velocities along the tubular network deeper in the fibre under our conditions (19 ± 1°C) ranged between 8 and 11 µm ms(-1) and 5 to 9 µm ms(-1), respectively, using different methods of estimation. The longitudinal propagation of APs appeared to be markedly faster closer to the edge of the fibre, in agreement with the presence of dense longitudinal connections immediately below the surface of the fibre and more sparse connections at deeper planes within the fibre. During long trains of closely spaced field pulses the AP-elicited Ca(2+) releases became non-synchronous along the fibre axis. This is most likely caused by local tubular K(+) accumulation that produces local depolarization and local slowing of AP propagation. Longitudinally propagating APs may reduce such inhomogeneities by exciting areas of delayed AP onset. Clearly, the longitudinal tubular pathways within the fibre for excitation are used as a safety mechanism in situations where a local depolarization obstructs immediate excitation from the sarcolemma. Results obtained from this study also provide an explanation for the pattern of contractures observed in rippling muscle disease.

Qualitatively different cross-bridge attachments in fast and slow muscle fiber types.

Contractile properties differ between skeletal, cardiac and smooth muscles as well as between various skeletal muscle fiber types. This functional diversity is thought to be mainly related to different speeds of myosin head pulling cycles, with the molecular mechanism of force generation being essentially the same. In this study, force-generating attachments of myosin heads were investigated by applying small perturbations of myosin head pulling cycles in stepwise stretch experiments on skeletal muscle fibers of different type. Slow fibers (frog tonic and rat slow-twitch) exhibited only a 'slow-type' of myosin head attachment over the entire activation range, while fast fibers (frog and rat fast-twitch) displayed a 'slow-type' of myosin head attachment at low levels of activation, and an up to 30-times faster type at high levels of activation. These observations indicate that there are qualitative differences between the mechanisms of myosin head attachment in slow and fast vertebrate skeletal muscle fibers.

Rapid Ca2+ flux through the transverse tubular membrane, activated by individual action potentials in mammalian skeletal muscle.

Periods of low frequency stimulation are known to increase the net Ca(2+) uptake in skeletal muscle but the mechanism responsible for this Ca(2+) entry is not known. In this study a novel high-resolution fluorescence microscopy approach allowed the detection of an action potential-induced Ca(2+) flux across the tubular (t-) system of rat extensor digitorum longus muscle fibres that appears to be responsible for the net uptake of Ca(2+) in working muscle. Action potentials were triggered in the t-system of mechanically skinned fibres from rat by brief field stimulation and t-system [Ca(2+)] ([Ca(2+)](t-sys)) and cytoplasmic [Ca(2+)] ([Ca(2+)](cyto)) were simultaneously resolved on a confocal microscope. When initial [Ca(2+)](t-sys) was > or = 0.2 mM a Ca(2+) flux from t-system to the cytoplasm was observed following a single action potential. The action potential-induced Ca(2+) flux and associated t-system Ca(2+) permeability decayed exponentially and displayed inactivation characteristics such that further Ca(2+) entry across the t-system could not be observed after 2-3 action potentials at 10 Hz stimulation rate. When [Ca(2+)](t-sys) was closer to 0.1 mM, a transient rise in [Ca(2+)](t-sys) was observed almost concurrently with the increase in [Ca(2+)](cyto) following the action potential. The change in direction of Ca(2+) flux was consistent with changes in the direction of the driving force for Ca(2+). This is the first demonstration of a rapid t-system Ca(2+) flux associated with a single action potential in mammalian skeletal muscle. The properties of this channel are inconsistent with a flux through the L-type Ca(2+) channel suggesting that an as yet unidentified t-system protein is conducting this current. This action potential-activated Ca(2+) flux provides an explanation for the previously described Ca(2+) entry and accumulation observed with prolonged, intermittent muscle activity.

Effect of temperature-induced reactive oxygen species production on excitation-contraction coupling in mammalian skeletal muscle.

1. Here we review evidence obtained recently by us indicating that the poor longevity of isolated mammalian skeletal muscle preparations at temperatures in the normal physiological range is related to the increased production of reactive oxygen species (ROS) in the resting muscle. 2. Temperature-induced ROS production increases markedly above 32 degrees C in isolated, resting skeletal muscle and is associated with the gradual and irreversible functional deterioration of the muscle. 3. The majority of the temperature-induced muscle ROS originates in the mitochondria and acts on various sites involved in excitation-contraction coupling.

O2(*-) production at 37 degrees C plays a critical role in depressing tetanic force of isolated rat and mouse skeletal muscle.

To find out whether the decrease in muscle performance of isolated mammalian skeletal muscle associated with the increase in temperature toward physiological levels is related to the increase in muscle superoxide (O(2)(*-)) production, O(2)(*-) released extracellularly by intact isolated rat and mouse extensor digitorum longus (EDL) muscles was measured at 22, 32, and 37 degrees C in Krebs-Ringer solution, and tetanic force was measured in both preparations at 22 and 37 degrees C under the same conditions. The rate of O(2)(*-) production increased marginally when the temperature was increased from 22 to 32 degrees C, but increased fivefold when the temperature was increased from 22 to 37 degrees C in both rat and mouse preparations. This increase was accompanied by a marked decrease in tetanic force after 30 min incubation at 37 degrees C in both rat and mouse EDL muscles. Tetanic force remained largely depressed after return to 22 degrees C for up to 120 min. The specific maximum Ca(2+)-activated force measured in mechanically skinned fibers after the temperature treatment was markedly depressed in mouse fibers but was not significantly depressed in rat muscle fibers. The resting membrane and intracellular action potentials were, however, significantly affected by the temperature treatment in the rat fibers. The effects of the temperature treatment on tetanic force, maximum Ca(2+)-activated force, and membrane potential were largely prevented by 1 mM Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl), a membrane-permeable superoxide dismutase mimetic, indicating that the increased O(2)(*-) production at physiological temperatures is largely responsible for the observed depression in tetanic force at 37 degrees C by affecting the contractile apparatus and plasma membrane.

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.

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.

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.

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.