D. George Stephenson

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

Examination of store-operated Ca2+ entry (SOC) in single, mechanically skinned skeletal muscle cells by confocal microscopy shows that the inositol 1,4,5-trisphosphate (IP3) receptor acts as a sarcoplasmic reticulum [Ca2+] sensor and mediates SOC by physical coupling without playing a key role in Ca2+ 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 IP3 receptors in skeletal muscle, the role of which with respect to Ca2+ homeostasis was ill defined until now.

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 [Ca2+] 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 [Ca2+] to ∼1 μm 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 Ca2+ release channels in the sarcoplasmic reticulum. This uncoupling showed a steep [Ca2+] dependency, with 50% uncoupling at ∼1.9 μm Ca2+. The uncoupling occurring with 2 μm Ca2+ was largely prevented by the calpain inhibitor leupeptin (1 mm). Raising the cytoplasmic [Ca2+] above 1 μm also caused an irreversible decline in passive force production in stretched skinned fibres in a manner graded by [Ca2+], though at a much slower relative rate than loss of coupling. The progressive loss of passive force could be rapidly stopped by lowering [Ca2+] to 10 nm, and was almost completely inhibited by 1 mm leupeptin but not by 10 μm calpastatin. Muscle homogenates preactivated by Ca2+ exposure also evidently contained a diffusible factor that caused damage to passive force production in a Ca2+‐dependent manner. Western blotting showed that: (a) calpain‐3 was present in the skinned fibres and was activated by the Ca2+exposure, and (b) the Ca2+ exposure in stretched skinned fibres resulted in proteolysis of titin. We conclude that the disruption of EC coupling occurring at elevated levels of [Ca2+] is likely to be caused at least in part by Ca2+‐activated proteases, most likely by calpain‐3, though a role of calpain‐1 is not excluded.

Effect of saponin treatment on the sarcoplasmic reticulum of rat, cane toad and crustacean (yabby) skeletal muscle.

1 Mechanically skinned fibres from skeletal muscles of the rat, toad and yabby were used to investigate the effect of saponin treatment on sarcoplasmic reticulum (SR) Ca2+ loading properties. The SR was loaded submaximally under control conditions before and after treatment with saponin and SR Ca2+ was released with caffeine. 2 Treatment with 10 μg ml−1 saponin greatly reduced the SR Ca2+ loading ability of skinned fibres from the extensor digitorum longus muscle of the rat with a rate constant of 0.24 min−1. Saponin concentrations up to 150 μg ml−1 and increased exposure time up to 30 min did not further reduce the SR Ca2+ loading ability of the SR, which indicates that the inhibitory action of 10–150 μg ml−1 saponin is not dose dependent. The effect of saponin was also not dependent on the state of polarization of the transverse‐tubular system. 3 Treatment with saponin at concentrations up to 100 μg ml−1 for 30 min did not affect the Ca2+ loading ability of SR in skinned skeletal muscle fibres from the twitch portion of the toad iliofibularis muscle but SR Ca2+ loading ability decreased markedly with a time constant of 0.22 min−1 in the presence of 150 μg ml−1 saponin. 4 The saponin dependent increase in permeability could be reversed in both rat and toad fibres by short treatment with 6 μM Ruthenium Red, a potent SR Ca2+ channel blocker, suggesting that saponin does affect the SR Ca2+ channel properties in mammalian and anuran skeletal muscle. 5 Treatment of skinned fibres of long sarcomere length (> 6 μM) from the claw muscle of the yabby (a freshwater decapod crustacean) with 10 μg ml−1 saponin for 30 min abolished the ability of the SR to load Ca2+, indicating that saponin affects differently the SR from skeletal muscles of mammals, anurans and crustaceans. 6 It is concluded that at relatively low concentrations, saponin causes inhibition of the skeletal SR Ca2+ loading ability in a species dependent manner, probably by increasing the Ca2+ loss through SR Ca2+ release channels.

Fiber type populations and Ca2+-activation properties of single fibers in soleus muscles from SHR and WKY rats

Electrophoretic analyses of muscle proteins in whole muscle homogenates and single muscle fiber segments were used to examine myosin heavy chain (MHC) and myosin light chain 2 (MLC2) isoform composition and fiber type populations in soleus muscles from spontaneously hypertensive rats (SHRs) and their age-matched normotensive controls [Wistar-Kyoto (WKY) rats], at three stages in the development of high blood pressure (4 wk, 16 wk, and 24 wk of age). Demembranated (chemically skinned with 2% Triton X-100), single fiber preparations were used to determine the maximum Ca2+-activated force per cross-sectional area, calcium sensitivity, and degree of cooperativity of the contractile apparatus and Ca2+-regulatory system with respect to Ca2+. The results show that, at all ages examined, 1) SHR soleus contained a lower proportion of MHCI and MLC2 slow (MLC2s) and a higher proportion of MHCIIa, MHCIId/x, and MLC2 fast (MLC2f ) isoforms than the age-matched controls; 2) random dissection of single fibers from SHR and WKY soleus produced four populations of fibers: type I (expressing MHCI), type IIA (expressing MHCIIa), hybrid type I+IIA (coexpressing MHCI and MHCIIa), and hybrid type IIA+IID (coexpressing MHCIIa and MHCIId/x); and 3) single fiber dissection from SHR soleus yielded a lower proportion of type I fibers, a higher proportion of fast-twitch fibers (types IIA and IIA+IID), and a higher proportion of hybrid fibers (types I+IIA and IIA+IID) than the homologous muscles from the age-matched WKY rats. Because the presence of hybrid fibers is viewed as a marker of muscle transformation, these data suggest that SHR soleus undergoes transformation well into adulthood. Our data show also that, for a given fiber type, there are no significant differences between SHR and WKY soleus muscles with respect to any of the Ca2+-activation properties examined. This finding indicates that the lower specific tensions reported in the literature for SHR soleus muscles are not due to strain- or hypertension-related differences in the function of the contractile apparatus or regulatory system.

Reversible changes in Ca2+-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 °C) led to reversible alterations of the contractile activation properties. These properties were studied using the mechanically skinned fibre preparation activated in Ca2+‐buffered solutions. The maximum Ca2+‐activated force (maximum force per cross‐sectional area) and the steepness of force‐pCa (‐log10[Ca2+]) curves as measured by the Hill coefficient (nH) reversibly decreased by factors of 8 and 2.5, respectively, when the EDL muscle was treated at 43 °C for 30 min and 5 and 2.8, respectively, with treatment at 46 °C for 5 min. Treatment at 47 °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 [Ca2+] 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 (O2−) production in muscle (apparent rate of O2− production at room temperature, 0.055 ± 0.008 nmol min−1 (g wet weight)−1; and following treatment to 46 °C for 5 min, 1.8 ± 0.2 nmol min−1 (g wet weight)−1) because 20 mm Tiron, a membrane‐permeant O2− scavenger, was able to markedly suppress the net rate of O2− 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 Ca2+ as the temperature increases in the upper physiological range through an increase in O2− production.

Effects of membrane cholesterol manipulation on excitation‐contraction coupling in skeletal muscle of the toad

1 Single mechanically skinned fibres and intact bundles of fibres from the twitch region of the iliofibularis muscle of cane toads were used to investigate the effects of membrane cholesterol manipulation on excitation‐contraction (E‐C) coupling. The cholesterol content of membranes was manipulated with methyl‐β‐cyclodextrin (MβCD). 2 In mechanically skinned fibres, depletion of membrane cholesterol with MβCD caused a dose‐ and time‐dependent decrease in transverse tubular (t)‐system depolarization‐induced force responses (TSDIFRs). TSDIFRs were completely abolished within 2 min in the presence of 10 mm MβCD but were not affected after 2 min in the presence of a 10 mm MβCD‐1 mm cholesterol complex. There was a very steep dependence between the change in TSDIFRs and the MβCD : cholesterol ratio at 10 mm MβCD, indicating that the inhibitory effect of MβCD was due to membrane cholesterol depletion and not to a pharmacological effect of the agent. Tetanic responses in bundles of intact fibres were abolished after 3‐4 h in the presence of 10 mm MβCD. 3 The duration of TSDIFRs increased markedly soon (< 2 min) after application of 10 mm MβCD and 10 mm MβCD‐cholesterol complexes, but the Ca2+ activation properties of the contractile apparatus were minimally affected by 10 mm MβCD. The Ca2+ handling abilities of the sarcoplasmic reticulum appeared to be modified after 10 min exposure to 10 mm MβCD. 4 Confocal laser scanning microscopy revealed that the integrity of the t‐system was not compromised by either intra‐ or extracellular application of 10 mm MβCD and that a large [Ca2+] gradient was maintained across the t‐system. 5 Membrane cholesterol depletion caused rapid depolarization of the polarized t‐system as shown independently by spontaneous TSDIFRs induced by MβCD and by changes in the fluorescence intensity of an anionic potentiometric dye (DiBAC4(3)) in the presence of MβCD. This rapid depolarization of the t‐system by cholesterol depletion was not prevented by blocking the Na+ channels with TTX (10 μm) or the L‐type Ca2+ channels with Co2+ (5 mm). 6 The results demonstrate that cholesterol is important for maintaining the functional integrity of the t‐system and sarcoplasmic reticulum, probably by having specific effects on different membrane proteins that may be directly or indirectly involved in E‐C coupling.

The effect of 2,5-di-(tert-butyl)-1,4-hydroquinone on force responses and the contractile apparatus in mechanically skinned muscle fibres of the rat and toad.

SummaryIn 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.

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‐sysVol) 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‐sysVol 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‐β‐cyclodextrin but did not change as the sarcomere length was changed from 1.93 to 3.30 μm. There was also an increase by 30 % and a decrease by 25 % in t‐sysVol 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‐sysVol 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.

Effect of clenbuterol on sarcoplasmic reticulum function in single skinned mammalian skeletal muscle fibers

We examined the effect of the beta2-agonist clenbuterol (50 microM) on depolarization-induced force responses and sarcoplasmic reticulum (SR) function in muscle fibers of the rat (Rattus norvegicus; killed by halothane overdose) that had been mechanically skinned, rendering the beta2-agonist pathway inoperable. Clenbuterol decreased the peak of depolarization-induced force responses in the extensor digitorum longus (EDL) and soleus fibers to 77.2 +/- 9.0 and 55.6 +/- 5.4%, respectively, of controls. The soleus fibers did not recover. Clenbuterol significantly and reversibly reduced SR Ca2+ loading in EDL and soleus fibers to 81.5 +/- 2.8 and 78.7 +/- 4.0%, respectively, of controls. Clenbuterol also produced an approximately 25% increase in passive leak of Ca2+ from the SR of the EDL and soleus fibers. These results indicate that clenbuterol has direct effects on fast- and slow-twitch skeletal muscle, in the absence of the beta2-agonist pathway. The increased Ca2+ leak in the triad region may lead to excitation-contraction coupling damage in the soleus fibers and could also contribute to the anabolic effect of clenbuterol in vivo.We examined the effect of the β2-agonist clenbuterol (50 μM) on depolarization-induced force responses and sarcoplasmic reticulum (SR) function in muscle fibers of the rat ( Rattus norvegicus; killed by halothane overdose) that had been mechanically skinned, rendering the β2-agonist pathway inoperable. Clenbuterol decreased the peak of depolarization-induced force responses in the extensor digitorum longus (EDL) and soleus fibers to 77.2 ± 9.0 and 55.6 ± 5.4%, respectively, of controls. The soleus fibers did not recover. Clenbuterol significantly and reversibly reduced SR Ca2+loading in EDL and soleus fibers to 81.5 ± 2.8 and 78.7 ± 4.0%, respectively, of controls. Clenbuterol also produced an ∼25% increase in passive leak of Ca2+ from the SR of the EDL and soleus fibers. These results indicate that clenbuterol has direct effects on fast- and slow-twitch skeletal muscle, in the absence of the β2-agonist pathway. The increased Ca2+ leak in the triad region may lead to excitation-contraction coupling damage in the soleus fibers and could also contribute to the anabolic effect of clenbuterol in vivo.

Analysis of Ca2+ and Sr2+ activation characteristics in skinned muscle fibre preparations with different proportions of myofibrillar isoforms.

SummaryTo 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.