Muscle RANK is a key regulator of Ca2+ storage, SERCA activity, and function of fast-twitch skeletal muscles.

Receptor-activator of nuclear factor-κB (RANK), its ligand RANKL, and the soluble decoy receptor osteoprotegerin are the key regulators of osteoclast differentiation and bone remodeling. Here we show that RANK is also expressed in fully differentiated myotubes and skeletal muscle. Muscle RANK deletion has inotropic effects in denervated, but not in sham, extensor digitorum longus (EDL) muscles preventing the loss of maximum specific force while promoting muscle atrophy, fatigability, and increased proportion of fast-twitch fibers. In denervated EDL muscles, RANK deletion markedly increased stromal interaction molecule 1 content, a Ca(2+)sensor, and altered activity of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) modulating Ca(2+)storage. Muscle RANK deletion had no significant effects on the sham or denervated slow-twitch soleus muscles. These data identify a novel role for RANK as a key regulator of Ca(2+)storage and SERCA activity, ultimately affecting denervated skeletal muscle function.

New method for determining total calcium content in tissue applied to skeletal muscle with and without calsequestrin.

We describe a new method for determining the concentration of total Ca in whole skeletal muscle samples ([CaT]WM in units of mmoles/kg wet weight) using the Ca-dependent UV absorbance spectra of the Ca chelator BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). Muscle tissue was homogenized in a solution containing 0.15 mM BAPTA and 0.5% sodium dodecyl sulfate (to permeabilize membranes and denature proteins) and then centrifuged. The solution volume was adjusted so that BAPTA captured essentially all of the Ca. [CaT]WM was obtained with Beer's law from the absorbance change produced by adding 1 mM EGTA to capture Ca from BAPTA. Results from mouse, rat, and frog muscles were reasonably consistent with results obtained using other methods for estimating total [Ca] in whole muscles and in single muscle fibers. Results with external Ca removed before determining [CaT]WM indicate that most of the Ca was intracellular, indicative of a lack of bound Ca in the extracellular space. In both fast-twitch (extensor digitorum longus, EDL) and slow-twitch (soleus) muscles from mice, [CaT]WM increased approximately linearly with decreasing muscle weight, increasing approximately twofold with a twofold decrease in muscle weight. This suggests that the Ca concentration of smaller muscles might be increased relative to that in larger muscles, thereby increasing the specific force to compensate for the smaller mass. Knocking out the high capacity Ca-binding protein calsequestrin (CSQ) did not significantly reduce [CaT]WM in mouse EDL or soleus muscle. However, in EDL muscles lacking CSQ, muscle weights were significantly lower than in wild-type (WT) muscles and the values of [CaT]WM were, on average, about half the expected WT values, taking into account the above [CaT]WM versus muscle weight relationship. Because greater reductions in [CaT]WM would be predicted in both muscle types, we hypothesize that there is a substantial increase in Ca bound to other sites in the CSQ knockout muscles.

Calcium buffering properties of sarcoplasmic reticulum and calcium-induced Ca(2+) release during the quasi-steady level of release in twitch fibers from frog skeletal muscle.

Experiments were performed to characterize the properties of the intrinsic Ca(2+) buffers in the sarcoplasmic reticulum (SR) of cut fibers from frog twitch muscle. The concentrations of total and free calcium ions within the SR ([Ca(T)](SR) and [Ca(2+)](SR)) were measured, respectively, with the EGTA/phenol red method and tetramethylmurexide (a low affinity Ca(2+) indicator). Results indicate SR Ca(2+) buffering was consistent with a single cooperative-binding component or a combination of a cooperative-binding component and a linear binding component accounting for 20% or less of the bound Ca(2+). Under the assumption of a single cooperative-binding component, the most likely resting values of [Ca(2+)](SR) and [Ca(T)](SR) are 0.67 and 17.1 mM, respectively, and the dissociation constant, Hill coefficient, and concentration of the Ca-binding sites are 0.78 mM, 3.0, and 44 mM, respectively. This information can be used to calculate a variable proportional to the Ca(2+) permeability of the SR, namely d[Ca(T)](SR)/dt ÷ [Ca(2+)](SR) (denoted release permeability), in experiments in which only [Ca(T)](SR) or [Ca(2+)](SR) is measured. In response to a voltage-clamp step to -20 mV at 15°C, the release permeability reaches an early peak followed by a rapid decline to a quasi-steady level that lasts ~50 ms, followed by a slower decline during which the release permeability decreases by at least threefold. During the quasi-steady level of release, the release amplitude is 3.3-fold greater than expected from voltage activation alone, a result consistent with the recruitment by Ca-induced Ca(2+) release of 2.3 SR Ca(2+) release channels neighboring each channel activated by its associated voltage sensor. Release permeability at -60 mV increases as [Ca(T)](SR) decreases from its resting physiological level to ~0.1 of this level. This result argues against a release termination mechanism proposed in mammalian muscle fibers in which a luminal sensor of [Ca(2+)](SR) inhibits release when [Ca(T)](SR) declines to a low level.

The concentration of free Ca(2+) in the sarcoplasmic reticulum of frog cut twitch skeletal muscle fibers estimated with tetramethylmurexide.

One aim of this article was to determine the resting concentration of free Ca(2+) in the sarcoplasmic reticulum (SR) of frog cut skeletal muscle fibers ([Ca(2+)](SR,R)) using the calcium absorbance indicator dye tetramethylmurexide (TMX). Another was to determine the ratio of [Ca(2+)](SR,R) to TMX's apparent dissociation constant for Ca(2+) (K(app)) in order to establish the capability of monitoring [Ca(2+)](SR)(t) during SR Ca(2+) release - a signal needed to determine the Ca(2+) permeability of the SR. To reveal the properties of TMX in the SR, the surface membrane was rapidly permeabilized with saponin to rapidly dissipate myoplasmic TMX. Results indicated that the concentration of Ca-free TMX in the SR was 2.8-fold greater than that in the myoplasm apparently due to binding of TMX to sites in the SR. Taking into account that such binding might influence K(app) as well as a dependence of K(app) on TMX concentration, the results indicate an average [Ca(2+)](SR,R) ranging from 0.43 to 1.70mM. The ratio [Ca(2+)](SR,R)/K(app) averaged 0.256, a relatively low value which should not depend on factors influencing K(app). As a result, the time course of [Ca(2+)](SR)(t) in response to electrical stimulation is well determined by, and approximately linearly related to, the active TMX absorbance signal.

Role of calsequestrin evaluated from changes in free and total calcium concentrations in the sarcoplasmic reticulum of frog cut skeletal muscle fibres.

Calsequestrin is a large-capacity Ca-binding protein located in the terminal cisternae of sarcoplasmic reticulum (SR) suggesting a role as a buffer of the concentration of free Ca in the SR ([Ca2+](SR)) serving to maintain the driving force for SR Ca2+ release. Essentially all of the functional studies on calsequestrin to date have been carried out on purified calsequestrin or on disrupted muscle preparations such as terminal cisternae vesicles. To obtain information about calsequestrin's properties during physiological SR Ca2+ release, experiments were carried out on frog cut skeletal muscle fibres using two optical methods. One - the EGTA-phenol red method - monitored the content of total Ca in the SR ([Ca(T)](SR)) and the other used the low affinity Ca indicator tetramethylmurexide (TMX) to monitor the concentration of free Ca in the SR. Both methods relied on a large concentration of the Ca buffer EGTA (20 mM), in the latter case to greatly reduce the increase in myoplasmic [Ca2+] caused by SR Ca2+ release thereby almost eliminating the myoplasmic component of the TMX signal. By releasing almost all of the SR Ca, these optical signals provided information about [Ca(T)](SR) versus [Ca2+](SR) as [Ca2+](SR) varied from its resting level ([Ca2+](SR,R)) to near zero. Since almost all of the Ca in the SR is bound to calsequestrin, this information closely resembles the binding curve of the Ca-calsequestrin reaction. Calcium binding to calsequestrin was found to be cooperative (estimated Hill coefficient = 2.95) and to have a very high capacity (at the start of Ca2+ release, 23 times more Ca was estimated to initiate from calsequestrin as opposed to the pool of free Ca in the SR). The latter result contrasts with an earlier report that only approximately 25% of released Ca2+ comes from calsequestrin and approximately 75% comes from the free pool. The value of [Ca2+](SR,R) was close to the K(D) for calsequestrin, which has a value near 1 mm in in vitro studies. Other evidence indicates that [Ca2+](SR,R) is near 1 mM in cut fibres. These results along with the known rapid kinetics of the Ca-calsequestrin binding reaction indicate that calsequestrin's properties are optimized to buffer [Ca2+](SR) during rapid, physiological SR Ca2+ release. Although the results do not entirely rule out a more active role in the excitation-contraction coupling process, they do indicate that passive buffering of [Ca2+](SR) is a very important function of calsequestrin.

Recruitment of Ca(2+) release channels by calcium-induced Ca(2+) release does not appear to occur in isolated Ca(2+) release sites in frog skeletal muscle.

Ca(2+) release from the sarcoplasmic reticulum (SR) in skeletal muscle in response to small depolarisations (e.g. to -60 mV) should be the sum of release from many isolated Ca(2+) release sites. Each site has one SR Ca(2+) release channel activated by its associated T-tubular voltage sensor. The aim of this study was to evaluate whether it also includes neighbouring Ca(2+) release channels activated by Ca-induced Ca(2+) release (CICR). Ca(2+) release in frog cut muscle fibres was estimated with the EGTA/phenol red method. The fraction of SR Ca content ([Ca(SR)]) released by a 400 ms pulse to -60 mV (denoted f(Ca)) provided a measure of the average Ca(2+) permeability of the SR associated with the pulse. In control experiments, f(Ca) was approximately constant when [Ca(SR)] was 1500-3000 microM (plateau region) and then increased as [Ca(SR)] decreased, reaching a peak when [Ca(SR)] was 300-500 microM that was 4.8 times larger on average than the plateau value. With 8 mM of the fast Ca(2+) buffer BAPTA in the internal solution, f(Ca) was 5.0-5.3 times larger on average than the plateau value obtained before adding BAPTA when [Ca(SR)] was 300-500 microM. In support of earlier results, 8 mM BAPTA did not affect Ca(2+) release in the plateau region. At intermediate values of [Ca(SR)], BAPTA resulted in a small, if any, increase in f(Ca), presumably by decreasing Ca inactivation of Ca(2+) release. Since BAPTA never decreased f(Ca), the results indicate that neighbouring channels are not activated by CICR with small depolarisations when [Ca(SR)] is 300-3000 microM.

Extra activation component of calcium release in frog muscle fibres.

In addition to activating more Ca(2+) release sites via voltage sensors in the t-tubular membranes, it has been proposed that more depolarised voltages enhance activation of Ca(2+) release channels via a voltage-dependent increase in Ca-induced Ca(2+) release (CICR). To test this, release permeability signals in response to voltage-clamp pulses to two voltages, -60 and -45 mV, were compared when Delta[Ca(2+)] was decreased in two kinds of experiments. (1) Addition of 8 mM of the fast Ca(2+) buffer BAPTA to the internal solution decreased release permeability at -45 mV by > 2-fold and did not significantly affect Ca(2+) release at -60 mV. Although some of this decrease may have been due to a decrease in voltage activation at -45 mV - as assessed from measurements of intramembranous charge movement - the results do tend to support a Ca-dependent enhancement with greater depolarisations. (2) Decreasing SR (sarcoplasmic reticulum) Ca content ([Ca(SR)]) should decrease the Ca(2+) flux through an open channel and thereby Delta[Ca(2+)]. Decreasing [Ca(SR)] from > 1000 microM (the physiological range) to < 200 microM decreased release permeability at -45 mV relative to that at -60 mV by > 6-fold, an effect shown to be reversible and not attributable to a decrease in voltage activation at -45 mV. These results indicate a Ca-dependent triggering of Ca(2+) release at more depolarised voltages in addition to that expected by voltage control alone. The enhanced release probably involves CICR and appears to involve another positive feedback mechanism in which Ca(2+) release speeds up the activation of voltage sensors.

Calcium release and intramembranous charge movement in frog skeletal muscle fibres with reduced (< 250 microM) calcium content.

It is generally accepted that activation of voltage sensors in the T-tubular membranes is a critical step of excitation-contraction coupling in skeletal muscle. The purpose of this study was to evaluate further whether the Qgamma component (delayed 'hump' component) of the intramembranous charge movement current (I(cm)) results from movement of these voltage sensors. Ca2+ release and I(cm) were measured in voltage-clamped frog cut fibres mounted in a double Vaseline-gap chamber. In order to reduce effects of Ca2+ feedback mechanisms, the calcium content of the sarcoplasmic reticulum (SR) during rest was reduced to < 250 microM (referred to volume of myoplasm) and maintained approximately constant. The early (Qbeta) and Qgamma components of charge movement were estimated by fitting the sum of two Boltzmann functions to the total steady-state intramembranous charge vs. voltage data. The average voltage steepness factor (k) and half-maximal voltage (V-) for Qgamma were 4.3 and -57.4 mV (n = 6), respectively. The SR membrane permeability for Ca2+ release was assessed when a constant amount of calcium remained in the SR (usually about 60 microM). A single Boltzmann function fitted to these data gave values on average for k and V- of 4.7 and -45.3 mV, respectively. The similarity of the values of k for Qgamma and Ca2+ release supports the idea that Qgamma reflects movement of voltage sensors for Ca2+ release. The greater value of V- for Ca2+ release compared to Qgamma is consistent with multi-state models of the voltage sensor involving movement of Qgamma charge during non-activating transitions.