Osmotic Properties of the Sealed Tubular System of Toad and Rat Skeletal Muscle

A method was developed that allows conversion of changes in maximum Ca2+-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 Ca2+ from the sealed t-system of toad fibers occurred, presumably through either stretch-activated cationic channels or store-operated Ca2+ 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

Regulation of Ca2+ Sparks by Ca2+ and Mg2+ in Mammalian and Amphibian Muscle. An RyR Isoform-specific Role in Excitation–Contraction Coupling?

Ca2+ and Mg2+ are important mediators and regulators of intracellular Ca2+ signaling in muscle. The effects of changes of cytosolic [Ca2+] or [Mg2+] on elementary Ca2+ release events were determined, as functions of concentration and time, in single fast-twitch permeabilized fibers of rat and frog. Ca2+ sparks were identified and their parameters measured in confocal images of fluo-4 fluorescence. Solutions with different [Ca2+] or [Mg2+] were rapidly exchanged while imaging. Faster and spatially homogeneous changes of [Ca2+] (reaching peaks >100 μM) were achieved by photolysing Ca NP-EGTA with laser flashes. In both species, incrementing cytosolic [Ca2+] caused a steady, nearly proportional increase in spark frequency, reversible upon [Ca2+] reduction. A greater change in spark frequency, usually transient, followed sudden increases in [Ca2+] after a lag of 100 ms or more. The nonlinearity, lag, and other features of this delayed effect suggest that it requires increase of [Ca2+] inside the SR. In the frog only, increases in cytosolic [Ca2+] often resulted, after a lag, in sparks that propagated transversally. An increase in [Mg2+] caused a fall of spark frequency, but with striking species differences. In the rat, but not the frog, sparks were observed at 4–40 mM [Mg2+]. Reducing [Mg2+] below 2 mM, which should enable the RyR channel's activation (CICR) site to bind Ca2+, caused progressive increase in spark frequency in the frog, but had no effect in the rat. Spark propagation and enhancement by sub-mM Mg2+ are hallmarks of CICR. Their absence in the rat suggests that CICR requires RyR3 para-junctional clusters, present only in the frog. The observed frequency of sparks corresponds to a channel open probability of 10−7 in the frog or 10−8 in the rat. Together with the failure of photorelease to induce activation directly, this indicates a basal inhibition of channels in situ. It is proposed that relief of this inhibition could be the mechanism by which increased SR load increases spark frequency

RyR1-targeted drug discovery pipeline integrating FRET-based high-throughput screening and human myofiber dynamic Ca2+ assays.

Elevated cytoplasmic [Ca2+] is characteristic in severe skeletal and cardiac myopathies, diabetes, and neurodegeneration, and partly results from increased Ca2+ leak from sarcoplasmic reticulum stores via dysregulated ryanodine receptor (RyR) channels. Consequently, RyR is recognized as a high-value target for drug discovery to treat such pathologies. Using a FRET-based high-throughput screening assay that we previously reported, we identified small-molecule compounds that modulate the skeletal muscle channel isoform (RyR1) interaction with calmodulin and FK506 binding protein 12.6. Two such compounds, chloroxine and myricetin, increase FRET and inhibit [3H]ryanodine binding to RyR1 at nanomolar Ca2+. Both compounds also decrease RyR1 Ca2+ leak in human skinned skeletal muscle fibers. Furthermore, we identified compound concentrations that reduced leak by &gt; 50% but only slightly affected Ca2+ release in excitation-contraction coupling, which is essential for normal muscle contraction. This report demonstrates a pipeline that effectively filters small-molecule RyR1 modulators towards clinical relevance

Ca2+ Sparks and Embers of Mammalian Muscle. Properties of the Sources

Ca2+ sparks of membrane-permeabilized rat muscle cells were analyzed to derive properties of their sources. Most events identified in longitudinal confocal line scans looked like sparks, but 23% (1,000 out of 4,300) were followed by long-lasting embers. Some were preceded by embers, and 48 were “lone embers.” Average spatial width was ∼2 μm in the rat and 1.5 μm in frog events in analogous solutions. Amplitudes were 33% smaller and rise times 50% greater in the rat. Differences were highly significant. The greater spatial width was not a consequence of greater open time of the rat source, and was greatest at the shortest rise times, suggesting a wider Ca2+ source. In the rat, but not the frog, spark width was greater in scans transversal to the fiber axis. These features suggested that rat spark sources were elongated transversally. Ca2+ release was calculated in averages of sparks with long embers. Release current during the averaged ember started at 3 or 7 pA (depending on assumptions), whereas in lone embers it was 0.7 or 1.3 pA, which suggests that embers that trail sparks start with five open channels. Analysis of a spark with leading ember yielded a current ratio ranging from 37 to 160 in spark and ember, as if 37–160 channels opened in the spark. In simulations, 25–60 pA of Ca2+ current exiting a point source was required to reproduce frog sparks. 130 pA, exiting a cylindric source of 3 μm, qualitatively reproduced rat sparks. In conclusion, sparks of rat muscle require a greater current than frog sparks, exiting a source elongated transversally to the fiber axis, constituted by 35–260 channels. Not infrequently, a few of those remain open and produce the trailing ember

The Changes in Ca2+ Sparks Associated with Measured Modifications of Intra-store Ca2+ Concentration in Skeletal Muscle

In cardiac muscle and amphibian skeletal muscle, the intracellular Ca2+ release that signals contractile activation proceeds by discrete local packets, which result in Ca2+ sparks. The remarkably stereotyped duration of these release events requires a robustly timed termination mechanism. In cardiac muscle the mechanism of spark termination appears to crucially involve depletion of Ca2+ in the lumen of the sarcoplasmic reticulum (SR), but in skeletal muscle, the mechanism is unknown. We used SEER (shifted excitation and emission ratioing of fluorescence) of SR-trapped mag-indo-1 and confocal imaging of fluorescence of cytosolic rhod-2 to image Ca2+ sparks while reversibly changing and measuring [Ca2+] in the SR ([Ca2+]SR) of membrane-permeabilized frog skeletal muscle cells. Sparks were collected in cells immersed in a solution promoting production of events at moderate frequency. Just after permeabilization, event frequency was zero, and in 10 minutes it reached close to a steady value. Controlled interventions modified [Ca2+]SR reversibly between a low value (299 μM on average in 10 experiments) and a high value (433 μM, a 45% average increase). This change increased sparks frequency by 93%, spatial width by 7%, rise time by 10%, and peak amplitude by 38% (provided that it was calculated in absolute terms, rather than normalized by resting fluorescence). The changes in event frequency and amplitude were statistically significant. The “strength” of the effect of [Ca2+]SR on frequency, quantified by decomposition of variance, was <6%. While the average change in [Ca2+]SR was limited, it reached up to 200% in individual fibers, without causing massive Ca2+ release or an increase of >3.5-fold in event frequency. Taken together with existing evidence that depletion is modest during Ca2+ sparks or release elicited by an action potential, the mild effects of [Ca2+]SR reported here do not support a major role of depletion in either the termination of sparks or the strong inactivation that terminates Ca2+ release at the global level in frog skeletal muscle

Concerted vs. Sequential. Two Activation Patterns of Vast Arrays of Intracellular Ca2+ Channels in Muscle

To signal cell responses, Ca2+ is released from storage through intracellular Ca2+ channels. Unlike most plasmalemmal channels, these are clustered in quasi-crystalline arrays, which should endow them with unique properties. Two distinct patterns of local activation of Ca2+ release were revealed in images of Ca2+ sparks in permeabilized cells of amphibian muscle. In the presence of sulfate, an anion that enters the SR and precipitates Ca2+, sparks became wider than in the conventional, glutamate-based solution. Some of these were “protoplatykurtic” (had a flat top from early on), suggesting an extensive array of channels that activate simultaneously. Under these conditions the rate of production of signal mass was roughly constant during the rise time of the spark and could be as high as 5 μm3 ms−1, consistent with a release current >50 pA since the beginning of the event. This pattern, called “concerted activation,” was observed also in rat muscle fibers. When sulfate was combined with a reduced cytosolic [Ca2+] (50 nM) these sparks coexisted (and interfered) with a sequential progression of channel opening, probably mediated by Ca2+-induced Ca2+ release (CICR). Sequential propagation, observed only in frogs, may require parajunctional channels, of RyR isoform β, which are absent in the rat. Concerted opening instead appears to be a property of RyR α in the amphibian and the homologous isoform 1 in the mammal

Calcium-dependent Inactivation Terminates Calcium Release in Skeletal Muscle of Amphibians

In skeletal muscle of amphibians, the cell-wide cytosolic release of calcium that enables contraction in response to an action potential appears to be built of Ca2+ sparks. The mechanism that rapidly terminates this release was investigated by studying the termination of Ca2+ release underlying sparks. In groups of thousands of sparks occurring spontaneously in membrane-permeabilized frog muscle cells a complex relationship was found between amplitude a and rise time T, which in sparks corresponds to the active time of the underlying Ca2+ release. This relationship included a range of T where a paradoxically decreased with increasing T. Three different methods were used to estimate Ca2+ release flux in groups of sparks of different T. Using every method, it was found that T and flux were inversely correlated, roughly inversely proportional. A simple model in which release sources were inactivated by cytosolic Ca2+ was able to explain the relationship. The predictive value of the model, evaluated by analyzing the variance of spark amplitude, was found to be high when allowance was made for the out-of-focus error contribution to the total variance. This contribution was estimated using a theory of confocal scanning (Ríos, E., N. Shirokova, W.G. Kirsch, G. Pizarro, M.D. Stern, H. Cheng, and A. González. Biophys. J. 2001. 80:169–183), which was confirmed in the present work by simulated line scanning of simulated sparks. Considering these results and other available evidence it is concluded that Ca2+-dependent inactivation, or CDI, provides the crucial mechanism for termination of sparks and cell-wide Ca2+ release in amphibians. Given the similarities in kinetics of release termination observed in cell-averaged records of amphibian and mammalian muscle, and in spite of differences in activation mechanisms, CDI is likely to play a central role in mammals as well. Trivially, an inverse proportionality between release flux and duration, in sparks or in global release of skeletal muscle, maintains constancy of the amount of released Ca2+

Human skeletal muscle plasmalemma alters its structure to change its Ca2+-handling following heavy-load resistance exercise

High-force eccentric exercise results in sustained increases in cytoplasmic Ca2+ levels ([Ca2+]cyto), which can cause damage to the muscle. Here we report that a heavy-load strength training bout greatly alters the structure of the membrane network inside the fibres, the tubular (t-) system, causing the loss of its predominantly transverse organization and an increase in vacuolation of its longitudinal tubules across adjacent sarcomeres. The transverse tubules and vacuoles displayed distinct Ca2+-handling properties. Both t-system components could take up Ca2+ from the cytoplasm but only transverse tubules supported store-operated Ca2+ entry. The retention of significant amounts of Ca2+ within vacuoles provides an effective mechanism to reduce the total content of Ca2+ within the fibre cytoplasm. We propose this ability can reduce or limit resistance exercise-induced, Ca2+-dependent damage to the fibre by the reduction of [Ca2+]cyto to help maintain fibre viability during the period associated with delayed onset muscle soreness