Mice with a malignant hyperthermia mutation (Y522S) in the ryanodine receptor (RyR1) display muscle contractures, rhabdomyolysis, and death in response to elevated environmental temperatures. We demonstrate that this mutation in RyR1 causes Ca(2+) leak, which drives increased generation of reactive nitrogen species (RNS). Subsequent S-nitrosylation of the mutant RyR1 increases its temperature sensitivity for activation, producing muscle contractures upon exposure to elevated temperatures. The Y522S mutation in humans is associated with central core disease. Many mitochondria in the muscle of heterozygous Y522S mice are swollen and misshapen. The mutant muscle displays decreased force production and increased mitochondrial lipid peroxidation with aging. Chronic treatment with N-acetylcysteine protects against mitochondrial oxidative damage and the decline in force generation. We propose a feed-forward cyclic mechanism that increases the temperature sensitivity of RyR1 activation and underlies heat stroke and sudden death. The cycle eventually produces a myopathy with damaged mitochondria.

Determination of the calcium spark amplitude distribution is of critical importance for understanding the nature of elementary calcium release events in striated muscle. In the present study we show, on general theoretical grounds, that calcium sparks, as observed in confocal line scan images, should have a nonmodal, monotonic decreasing amplitude distribution, regardless of whether the underlying events are stereotyped. To test this prediction we developed, implemented, and verified an automated computer algorithm for objective detection and measurement of calcium sparks in raw image data. When the sensitivity and reliability of the algorithm were set appropriately, we observed highly left-skewed or monotonic decreasing amplitude distributions in skeletal muscle cells and cardiomyocytes, confirming the theoretical predictions. The previously reported modal or Gaussian distributions of sparks detected by eye must therefore be the result of subjective detection bias against small amplitude events. In addition, we discuss possible situations when a modal distribution might be observed.

Puzzled by recent reports of differences in specific ligand binding to muscle Ca 2+ channels, we quantitatively compared the flux of Ca 2+ release from tile sarcoplasmic reticulum (SR) in skeletal muscle fibers of an amphibian (frog) and a mammal (rat), voltage clamped in a double Vaseline gap chamber. The determinations of release flux were carried out by the "removal" method and by measuring the rate of Ca 2+ binding to dyes in large excess over other Ca 2+ buffers. To have a more meaningful comparison, the effects of stretching the fibers, of rapid changes in temperature, and of changes in the Ca 2 § content of the SR were studied in both species. In both frogs and rats, the release flux had an early peak followed by fast relaxation to a lower sustained release. The peak and steady values of release flux, Rp and R~, were influenced little by stretching. Rp in frogs was 31 mM/s (SEM = 4, n = 24) and in rats 7 4-2 mM/s (n = 12). R~ was 9 + 1 and 3 4-0.7 mM/s in frogs and rats, respectively. Transverse (T) tubule area, estimated from capacitance measurements and norrealized to fiber volume, was greater in rats (0.61 + 0.04 ixm -1) than in frogs (0.48 4-0.04 ixm-l), as expected from the greater density of T tubuli. Total Ca in the SR was estimated as 3.4 ---0.6 and 1.9 + 0.3 mmol/liter myoplasmic water in frogs and rats. With the above figures, the steady release flux per unit area of T tubule was found to be fourfold greater in the frog, and the steady permeability of the junctional SR was about threefold greater. The ratio Rp/Rs was ~2 in rats at all voltages, whereas it was greater and steeply voltage dependent in frogs, going through a maximum of 6 at -40 mV, then decaying to ~3.5 at high voltage. Both Rp and R~ depended strongly on the temperature, but their ratio, and its voltage dependence, did not. Assuming that the peak of Ca 2+ release is contributed by release channels not in contact with voltage sensors, or not under their direct control, the greater ratio in frogs may correspond to the relative excess of Ca 2+ release channels over voltage sensors apparent in binding measurements. From the marked differences in voltage dependence of the ratio, as well as consideration of Ca2+-induced release models, we derive indications of fundamental differences in control mechanisms between mammalian and amphibian muscle.

Fluo‐3 fluorescence associated with Ca2+ release was recorded with confocal microscopy in single muscle fibres mechanically dissected from fast twitch muscle of rats or frogs, voltage clamped in a two Vaseline‐gap chamber. Interventions that elicited Ca2+ sparks in frog skeletal muscle (low voltage depolarizations, application of caffeine) generated in rat fibres images consistent with substantial release from triadic regions, but devoid of resolvable discrete events. Ca2+ sparks were never observed in adult rat fibres. In contrast, sparks of standard morphology were abundant in myotubes from embryonic mice. Depolarization‐induced gradients of fluorescence between triadic and surrounding regions (which are proportional to Ca2+ release flux) peaked at about 20 ms and then decayed to a steady level. Gradients were greater in frog fibres than in rat fibres. The ratio of peak over steady gradient (R) was steeply voltage dependent in frogs, reaching a maximum of 4.8 at −50 mV (n= 7). In rats, R had an essentially voltage‐independent value of 2.3 (n= 5). Ca2+‐induced Ca2+ release, resulting in concerted opening of several release channels, is thought to underlie Ca2+ sparks and the peak phase of release in frog skeletal muscle. A diffuse ‘small event’ release, similar to that observed in these rats, is also present in frogs and believed to be directly activated by voltage. The present results suggest that in these rat fibres there is little contribution by CICR to Ca2+ release triggered by depolarization, and a lack of concerted channel opening.

Overall, our findings reveal that excessive intracellular Ca(2+) signals and ROS generation link the initial sarcolemmal injury to mitochondrial dysfunctions. The latter possibly contribute to the loss of functional cardiac myocytes and heart failure in dystrophy. Understanding the sequence of events of dystrophic cell damage and the deleterious amplification systems involved, including several positive feed-back loops, may allow for a rational development of novel therapeutic strategies.

In the present study, the link between cellular metabolism and Ca2+ signalling was investigated in permeabilized mammalian skeletal muscle. Spontaneous events of Ca2+ release from the sarcoplasmic reticulum were detected with fluo‐3 and confocal scanning microscopy. Mitochondrial functions were monitored by measuring local changes in mitochondrial membrane potential (with the potential‐sensitive dye tetramethylrhodamine ethyl ester) and in mitochondrial [Ca2+] (with the Ca2+ indicator mag‐rhod‐2). Digital fluorescence imaging microscopy was used to quantify changes in the mitochondrial autofluorescence of NAD(P)H. When fibres were immersed in a solution without mitochondrial substrates, Ca2+ release events were readily observed. The addition of l‐glutamate or pyruvate reversibly decreased the frequency of Ca2+ release events and increased mitochondrial membrane potential and NAD(P)H production. Application of various mitochondrial inhibitors led to the loss of mitochondrial [Ca2+] and promoted spontaneous Ca2+ release from the sarcoplasmic reticulum. In many cases, the increase in the frequency of Ca2+ release events was not accompanied by a rise in global [Ca2+]i. Our results suggest that mitochondria exert a negative control over Ca2+ signalling in skeletal muscle by buffering Ca2+ near Ca2+ release channels.

An algorithm for the calculation of Ca2+ release flux underlying Ca2+ sparks (Blatter, L.A., J. Hüser, and E. Ríos. 1997. Proc. Natl. Acad. Sci. USA. 94:4176–4181) was modified and applied to sparks obtained by confocal microscopy in single frog skeletal muscle fibers, which were voltage clamped in a two-Vaseline gap chamber or permeabilized and immersed in fluo-3–containing internal solution. The performance of the algorithm was characterized on sparks obtained by simulation of fluorescence due to release of Ca2+ from a spherical source, in a homogeneous three-dimensional space that contained components representing cytoplasmic molecules and Ca2+ removal processes. Total release current, as well as source diameter and noise level, was varied in the simulations. Derived release flux or current, calculated by volume integration of the derived flux density, estimated quite closely the current used in the simulation, while full width at half magnitude of the derived release flux was a good monitor of source size only at diameters >0.7 μm. On an average of 157 sparks of amplitude >2 U resting fluorescence, located automatically in a representative voltage clamp experiment, the algorithm reported a release current of 16.9 pA, coming from a source of 0.5 μm, with an open time of 6.3 ms. Fewer sparks were obtained in permeabilized fibers, so that the algorithm had to be applied to individual sparks or averages of few events, which degraded its performance in comparable tests. The average current reported for 19 large sparks obtained in permeabilized fibers was 14.4 pA. A minimum estimate, derived from the rate of change of dye-bound Ca2+ concentration, was 8 pA. Such a current would require simultaneous opening of between 8 and 60 release channels with unitary Ca2+ currents of the level recorded in bilayer experiments. Real sparks differ from simulated ones mainly in having greater width. Correspondingly, the algorithm reported greater spatial extent of the source for real sparks. This may again indicate a multichannel origin of sparks, or could reflect limitations in spatial resolution.

In many types of muscle, intracellular Ca 2؉ release for contraction consists of brief Ca 2؉ sparks. Whether these result from the opening of one or many channels in the sarcoplasmic reticulum is not known. Examining massive numbers of sparks from frog skeletal muscle and evaluating their Ca 2؉ release current, we provide evidence that they are generated by multiple channels. A mode is demonstrated in the distribution of spark rise times in the presence of the channel activator caffeine. This finding contradicts expectations for single channels evolving reversibly, but not for channels in a group, which collectively could give rise to a stereotyped spark. The release channel agonists imperatoxin A, ryanodine, and bastadin 10 elicit fluorescence events that start with a spark, then decay to steady levels roughly proportional to the unitary conductances of 35%, 50%, and 100% that the agonists, respectively, promote in bilayer experiments. This correspondence indicates that the steady phase is produced by one open channel. Calculated Ca 2؉ release current decays 10-to 20-fold from spark to steady phase, which requires that six or more channels be open during the spark. (4, 5), and smooth muscle (6). Sparks are fundamental in health and disease (7), but their mechanism, especially whether one or many channels are involved in the spark generator or ''release unit,'' remains unclear (8-10). Answering this question, which pervades the field since its inception (2), will help understand how the channels are coaxed by their agonists (Ca, membrane voltage; reviewed in refs. 11 and 12) and restrained by their antagonists (Ca, Mg) to shape these events.Here we improve a technique for detection of massive numbers of sparks (13) Materials and MethodsExperiments were carried out at 17°C in cut skeletal muscle fibers from Rana pipiens semitendinosus muscle, stretched at 3-3.5 m͞sarcomere, either voltage-clamped in a two-Vaseline gap chamber or permeabilized and immersed in internal solution, on an inverted microscope. Adult frogs anaesthetized in 15% ethanol were killed by pithing. The external solution contained 10 mM Ca(CH 3 SO 3 ) 2 , 130 mM tetraethylammonium-CH 3 SO 3 , 5 mM Tris maleate, 1 mM 3,4 diaminopyridine, and 1 M tetrodotoxin. The internal solution contained 110 mM Cs-glutamate, 1 mM EGTA, 5 mM glucose, 5 mM Mg-ATP, 5 mM phosphocreatine, 10 mM Hepes, 0.2 mM fluo-3, with 100 nM free [Ca 2ϩ ] and 1.8 mM [Mg 2ϩ ]. For permeabilized cells the internal solution contained 0.05 mM fluo-3 and 0.34 mM [Mg 2ϩ ] and included 4% 10-kDa dextran. Solutions were adjusted to pH 7 and 270 mosmol͞kg. The scanning microscope (MRC 1000, Bio-Rad) was in fluo-3 configuration (14) using a 40ϫ, 1.2 numerical aperture water immersion objective (Zeiss). Images shown are of fluorescence determined at 2-ms intervals (4.3 ms in toxin experiments) along a parallel to the fiber axis. Fluorescence F(x,t) is presented normalized to its average F 0 (x) before the voltage pulse. Sparks are located on a spatially filtered version of...