Biallelic variants in PPP1R13L cause paediatric dilated cardiomyopathy.

Childhood dilated cardiomyopathy (DCM) is a leading cause of heart failure requiring cardiac transplantation and approximately 5% of cases result in sudden death. Knowledge of the underlying genetic cause can aid prognostication and clinical management and enables accurate recurrence risk counselling for the family. Here we used genomic sequencing to identify the causative genetic variant(s) in families with children affected by severe DCM. In an international collaborative effort facilitated by GeneMatcher, biallelic variants in PPP1R13L were identified in seven children with severe DCM from five unrelated families following exome or genome sequencing and inheritance-based variant filtering. PPP1R13L encodes inhibitor of apoptosis-stimulating protein of p53 protein (iASPP). In addition to roles in apoptosis, iASPP acts as a regulator of desmosomes and has been implicated in inflammatory pathways. DCM presented early (mean: 2 years 10 months; range: 3 months-9 years) and was progressive, resulting in death (n = 3) or transplant (n = 3), with one child currently awaiting transplant. Genomic sequencing technologies are valuable for the identification of novel and emerging candidate genes. Biallelic variants in PPP1R13L were previously reported in a single consanguineous family with paediatric DCM. The identification here of a further five families now provides sufficient evidence to support a robust gene-disease association between PPP1R13L and severe paediatric DCM. The PPP1R13L gene should be included in panel-based genetic testing for paediatric DCM.

[Maternal effect obscures adaptation to adverse environments and hinders divergence in Drosophila melanogaster].

Adaptation to contrasting environments can facilitate ecological divergence and sympatric speciation. Factors that influence the probability and tempo of these processes are poorly known. We performed an evolutionary experiment on Drosophila melanogaster in order to attain better understanding of adaptation dynamics and to model the initial steps of sympatric speciation. In our experiment, several populations are being cultured either on standard rich medium (RM) or on nutrient-deficient starch-based medium (SM). After 10 generations, experimental populations demonstrated unexpected changes in their fitness: on the starch medium, flies grown on RM (FRM) outcompeted those that were cultured on SM (FSM), while on the rich medium, FRM were outcompeted by FSM. That is, experimental populations demonstrated higher fitness on the foreign medium, but were outcompeted by the aliens on the one they had been accustomed to. To explain the paradox, we hypothesize that the observed low fitness of FSM on SM was due to maternal effect, or the "effect of starving mother". The FSM flies are probably better adapted to SM, but the phenotypic outcome of their adaptations is obscured because the females grown on the poor medium invest less in their offspring (for instance, they may produce nutrient-deficient eggs). Larvae hatched from such eggs develop successfully on the rich medium RM, but experience delayed growth and/or lower survival rate on the nutrient-deficient medium SM. To test the hypothesis, we measured the fitness of the flies FSM after culturing them for one generation on RM, in order to remove the assumed maternal effect. As expected, this time FSM demonstrated higher fitness on SM compared to control flies (FRM) and to FSM before the removal of the maternal effect. The results support the idea that non-adaptive phenotypic plasticity and maternal effects can mask adaptation to adverse environments and prohibit ecological divergence and speciation by allowing the migrants from favourable habitats to outcompete resident individuals in adverse ecotopes despite the possible presence of specific adaptations in the latter.

The significance of increased levels of end nitric oxide metabolites in blood serum of children with celiac disease.

OBJECTIVES: The aim of the study was to determine serum nitric oxide (NO) metabolites and correlate them to gluten-free diet (GFD) compliance, the parameters of morphological and functional condition of the small intestine in patients with celiac disease (CD) in a long-term follow-up. PATIENTS AND METHODS: The study included 35 children (age median 8.0 years) with previously diagnosed CD, among them 15 - on a strict GFD and 20 - on a semistrict GFD. 32 patients (age median 7.5 years) with functional abdominal pain were recruited as controls. Nitrites (NO(2)), nitrates (NO(3)), total content of nitrites and nitrates (NOx) in serum were determined using the Griess reaction. CD patients underwent small intestine morphometry and absorption D-xylose test. RESULTS: Compared to the controls, the CD patients on a strict GFD had no significant differences in NO(2), NO(3), NOx content, while in patients violating GFD these parameters were significantly higher. In comparison with the patients on a strict GFD, the patients on a semistrict GFD had higher median values of NO(3) (p = 0.002) and NOx (p = 0.0000). Serum NOx correlated negatively with the height of villi (p = 0.0001), D-xylose excretion (p = 0.0063), and correlated positively with the depth of crypts (p = 0.0082) and cellular density of villi stroma (p = 0.0067). CONCLUSION: CD patients not keeping to a strict GFD have an increased level of nitric oxide metabolites in serum. NOx content is associated with morphometric parameters of the small intestine that proves the participation of NO in the pathogenesis of histological changes in CD (Tab. 3, Ref. 39).

Duodenal mucosa in children with coeliac disease in catamnesis and varying compliance with the gluten-free diet.

OBJECTIVES: the aim of the study was to identify the characteristic morphometric data changes in the small intestine mucosa in response to varied compliance with gluten-free diet in children with coeliac disease. METHODS: 71 children (47 girls and 24 boys) aged 2.5 to 16.5 (median age=10.6) with coeliac disease diagnosed according to the previously revised ESPGHAN criteria were included in the study. The patients were divided into three groups: 1 - on strict GFD, 2 - on semistrict GFD, 3 - not on GFD. Quantitative morphometric data on biopsies of duodenal mucosa in each group were compared with each other. RESULTS: As compared with group 1, patients from group 2 had a lower villous height (Vh)/crypt depth (Cd) ratio, but a higher Cd, total cell density of lamina propria, plasma cells density in the villi and fibroblasts density in the space between the crypts. In patients from group 3, as compared with group 1, we found a significant reduction in Vh and Vh/Cd ratio, and a significant decrease in the number of goblet cells. At the same time they yielded an increase in Cd, number of intraepithelial lymphocytes (IEL), total cell density and plasma cells density throughout the lamina propria depth, number of macrophages and neutrophils in the villous lamina propria, and number of fibroblasts in the space between crypts. The differences were statistically significant. CONCLUSION: Quantitative morphometric parameters of small intestine mucosa allow revealing statistically significant differences between the groups of children with coeliac disease in response to varied compliance with GFD. This enables us to assess the dynamics of the pathological process and enhance our understanding of its nature, which is very important for improving the therapy and prognosis (Ref. 40).

[Features of mucosal lesions of stomach and duodenum in children with chronic gastroduodenitis with different microflora content].

OBJECTIVE: Our aim was to reveale the features of histological mucosa with different composition of the microflora of stomach and duodenum in children with chronic gastroduodenitis. METHODS: The study included 122 children with chronic gastroduodenitis from 5 to 17 years old. All patients underwent endoscopy with histology of biopsy specimens of gastric and duodenal mucosa in 94 patients and in 48 patients among them with morphometry of biological material. Identification of herpes simplex virus 1, 2 types, cytomegalovirus, human papilloma virus 16, 18 types, Helicobacterpylori was carried out using polymerase chain reaction in biopsies of gastroduodenal mucosa and gastric juice. RESULTS: Children with chronic gastroduodenitis depending on the identified microorganisms were divided into groups: 1st (n = 51)--without detectable microorganisms, 2nd (n = 28)--with associations of H. pylori and viruses, 3rd (n = 15)--only with viruses, 4th (n = 28)--with H. pylori. Thus, in patients with H. pylori inflammation was noted mainly in the antral mucosa and dystrophic changes--in the duodenal mucosa. In the presence of viruses the inflammation was expressed more in the fundal stomach and duodenum. When viral-bacterial associations were observed most pronounced inflammatory-dystrophic process was localized throughout the gastroduodenal mucosa. CONCLUSION: In children with chronic gastroduodenitis in the presence of bacterial, viral and mixed microflora in the stomach and duodenum identified characteristic histological features of the mucosa, manifested in a different localization and severity of inflammatory and dystrophic changes.

[Changes of epidermal growth factor level in blood serum, saliva and gastric juice in children with duodenal ulcer].

AIM: The aim of our study is to investigate EGF content in biological mediums in children with duodenum ulcer depending on phase of the disease and different variants of its course. MATERIALS AND METHODS: The present study was performed in Federal State Establishment "Nizhniy Novgorod Research Institute of Children Gastroenterology", Nizhniy Novgorod, Russia. 92 children, between the ages of 8 to 17, with duodenum ulcer were under observation. Endoscopy was performed by Pentax endoscope (FG-24V). EGF detection was performed in blood serum, gastric juice and saliva by ELISA method with Human EGF Kit, "Invitrogen", USA. RESULTS: The peculiarities of EGF level changes in human biological mediums, depending on phase of the disease. The highest EGF level was detected with acute peptic ulcer in the presence of ulcerous defects. EGF level increasing was marked out in the remission phaseas ulcerous defects healing, and it didn't reach normal values in gastric juice. EGF content changes in biological mediums were revealed with different variants of duodenum ulcer clinical course in children. The lowest EGF level was marked out in blood, saliva and gastric juice with unfavorable course of the disease (frequent relapses, cicatricial-ulcerous strains formation), which can serve as a prognostic factor.

[Content of epidermal growth factor in biosubstrates and macrophage count in defect healing in children and adolescents with duodenal ulcer disease].

The concentrations of epidermal growth factor (EGF) in saliva, gastric juice, and blood and those of macrophages in mucosal biopsy specimens were determined in children with duodenal ulcer disease. The maximum level of EGF was noted in the saliva. There were unidirectional positive changes in the levels of EGF and macrophages during ulcerative defect healing.

Pathways of abnormal stress-induced Ca2+ influx into dystrophic mdx cardiomyocytes.

In Duchenne muscular dystrophy, deficiency of the cytoskeletal protein dystrophin leads to well-described defects in skeletal muscle, but also to dilated cardiomyopathy, accounting for about 20% of the mortality. Mechanisms leading to cardiomyocyte cell death and cardiomyopathy are not well understood. One hypothesis suggests that the lack of dystrophin leads to membrane instability during mechanical stress and to activation of Ca2+ entry pathways. Using cardiomyocytes isolated from dystrophic mdx mice we dissected the contribution of various putative Ca2+ influx pathways with pharmacological tools. Cytosolic Ca2+ and Na+ signals as well as uptake of membrane impermeant compounds were monitored with fluorescent indicators using confocal microscopy and photometry. Membrane stress was applied as moderate osmotic challenges while membrane current was quantified using the whole-cell patch-clamp technique. Our findings suggest a major contribution of two primary Ca2+ influx pathways, stretch-activated membrane channels and short-lived microruptures. Furthermore, we found evidence for a secondary Ca2+ influx pathway, the Na+-Ca2+ exchange (NCX), which in cardiac muscle has a large transport capacity. After stress it contributes to Ca2+ entry in exchange for Na+ which had previously entered via primary stress-induced pathways, representing a previously not recognized mechanism contributing to subsequent cellular damage. This complexity needs to be considered when targeting abnormal Ca2+ influx as a treatment option for dystrophy.

[Absorption of fatty acids by the rat heart in arrhythmia].

Fifty-three white rat males were divided into 4 groups: intact, with arrhythmia, with arrhythmia treated with cordaron, with arrhythmia treated with cardiotron. The rats' diet was enriched with animal fats. Anesthesia was made with uretan (1,0-1,2 g/kg, i.p.). Arrhythmia was induced with akonitine (30-40 mg/kg, i.v.). The drugs were administered 12 and 25 days before arrhythmia induction which was registered on ECG at min 3, 5, 15 and 25 in 1, 11 and 111 standard leads. Total lipid fatty acids (TLFA) of plasma were detected in heart inflowing and outflowing blood with gas chromatography. Arrhythmia drastically changes qualitative characteristics of arterio-venous difference for fatty acids. Cordaron and cardiotron prevented arrhythmia in 63-75% cases. TLFA arterio-venous difference recovered by 55-69%. Cordaron and cardiotron may have a mediating mechanism of action on fatty acid absorption by the heart in normalization of normal heart rate in experimental arrhythmia.

[Efficacy of the new collection of herbs at stressful experimental sharp ulcer defects of the gastroduodenal zone].

As a result of experiments on white male rats after an immobilization stress that resulted in the formation of sharp ulcer defects in the gastroduodenal zone, it was established that the daily 5-day peroral administration of the collection of herbs (Korniozil) in the volume of 4-6 ml in equal dozes 3 times a day as 9-12% of a water-alcohol solution had a positive influence on the regeneration of the mucous coat of the stomach. The metabolism and structure of lipids in the mucous coat of the stomach as well as balance between activity of processes of peroxide lipid oxidations and functioning of antioxidant systems was simultaneously restored.

Beta-1,3-glucanase from unfertilized eggs of the sea urchin Strongylocentrotus intermedius. Comparison with beta-1,3-glucanases of marine and terrestrial mollusks.

beta-1,3-Glucanase (Lu) was isolated from unfertilized eggs of the sea urchin Strongylocentrotus intermedius. A comparative study of some properties of beta-1,3-glucanase Lu and beta-1,3-glucanases with different action types--endo-beta-1,3-glucanase from crystalline style of the marine mollusk Spisula sachalinensis (LIV) and exo-beta-1,3-glucanase from the terrestrial snail Eulota maakii (LII)--was performed. It was found that beta-1,3-glucanase Lu hydrolyzes laminaran with a high yield of glucose in the reaction products. The enzyme hydrolyzes substrates with retention of the glycosidic bond configuration, is able to cleave modified substrates, and exhibits transglycosylating activity. All properties of beta-1,3-glucanase from S. intermedius were more similar to those of the endo-beta-1,3-glucanase from the marine mollusk (LIV) than exo-beta-1,3-glucanase LII from the terrestrial snail. The differences in the effect of LIV and Lu on laminaran are probably related to the functions of beta-1,3-glucanase Lu from sea urchin eggs (which, in contrast to LIV, is not a digestive enzyme).

A preferred amplitude of calcium sparks in skeletal muscle.

In skeletal and cardiac muscle, calcium release from the sarcoplasmic reticulum, leading to contraction, often results in calcium sparks. Because sparks are recorded by confocal microscopy in line-scanning mode, their measured amplitude depends on their true amplitude and the position of the spark relative to the scanned line. We present a method to derive from measured amplitude histograms the actual distribution of spark amplitudes. The method worked well when tested on simulated distributions of experimental sparks. Applied to massive numbers of sparks imaged in frog skeletal muscle under voltage clamp in reference conditions, the method yielded either a decaying amplitude distribution (6 cells) or one with a central mode (5 cells). Caffeine at 0.5 or 1 mM reversibly enhanced this mode (5 cells) or induced its appearance (4 cells). The occurrence of a mode in the amplitude distribution was highly correlated with the presence of a mode in the distribution of spark rise times or in the joint distribution of rise times and spatial widths. If sparks were produced by individual Markovian release channels evolving reversibly, they should not have a preferred rise time or amplitude. Channel groups, instead, could cooperate allosterically or through their calcium sensitivity, and give rise to a stereotyped amplitude in their collective spark.

Fast imaging in two dimensions resolves extensive sources of Ca2+ sparks in frog skeletal muscle.

Ca2+ sparks were monitored by confocal laser-scanning microscopy of fluo-3 at video rates, in fast twitch muscle fibres, stimulated by exposure to caffeine and/or low [Mg2+]. Scanning was in two spatial dimensions ('2D') or 2D plus time, at 4 ms per image frame. Sparks were identified in 2D images of normalized fluorescence by an automatic procedure, which also evaluated the event's location and morphometric parameters. Most sparks were circular, but some were elongated, especially in caffeine. Separation of the spark from circular symmetry was quantified by its eccentricity (length/width - 1). In an internal solution with 0.4 mM [Mg2+], sparks (989 events in 4 cells) had amplitude 0.73, width 1.94 microm, length 2.12 microm and eccentricity not significantly different from 0. Upon application of 1 mM caffeine, length (of 2578 events in the same cells) increased significantly (by 0.41 microm, or 19 %), width increased by 0.18 microm (9 %) and amplitude decreased slightly. The eccentricity became significantly different from 0, and the sparks' long axis predominantly oriented parallel to the plane of the Z disks. More than 10 % of the events in caffeine had length greater than 4 microm, a relatively flat top, and a sharp termination at both ends of the major axis. Additionally, there was only a weak correlation between eccentricity and amplitude. These properties suggest that the elongated events are produced by simultaneous opening of multiple channels within a junction, rather than anisotropic diffusion of Ca2+ or random overlap of round sparks. Elongated events often increased in eccentricity early in their evolution. Then, most remained elongated during their rise and decay, while others spread spatially in the plane of the Z disks. In 1-2 % of the events, the centre of mass migrated in space, over time, at approximately 0.1 microm x ms(-1). These spatio-temporal features require the involvement of multiple release channels, at spatially resolvable locations. Because sources often spread over distances greater than 1 or 2 microm, and arrays of junctional elements (couplons) are at most 1.2 microm long, it must be possible for activation of release to propagate between neighbouring couplons, especially under the influence of caffeine and/or low [Mg2+].

Involvement of multiple intracellular release channels in calcium sparks of skeletal muscle.

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.

The spark and its ember: separately gated local components of Ca(2+) release in skeletal muscle.

Amplitude, spatial width, and rise time of Ca(2+) sparks were compared in frog fast-twitch muscle, in three conditions that alter activation of release channels by [Ca(2+)]. A total of approximately 17,000 sparks from 30 cells were evaluated. In cells under voltage clamp, caffeine (0.5 or 1 mM) increased average spark width by 28%, rise time by 18%, and amplitude by 7%. Increases in width were significant even among events of the same rise time. Spontaneous events recorded in permeabilized fibers with low internal [Mg(2+)] (0.4 mM), had width and rise times greater than in reference, and not significantly different than those in caffeine. The spark average in reference rides on a continuous fluorescence "ridge" and is continued by an "ember," a prolongation of width approximately 1 microm and amplitude <0.2, vanishing in approximately 100 ms. Ridge and ember were absent in caffeine and in permeabilized cells. Exposure of voltage-clamped cells to high internal [Mg(2+)] (7 mM) had effects opposite to caffeine, reducing spark width by 26% and amplitude by 27%. In high [Mg(2+)], the ember was visible in individual sparks as a prolongation of variable duration and amplitude up to 1.2. Based on simulations and calculation of Ca(2+) release flux from averaged sparks, the increase in spark width caused by caffeine was interpreted as evidence of an increase in radius of the release source-presumably by recruitment of additional channels. Conversely, spark narrowing suggests loss of contributing channels in high Mg(2+). Therefore, these changes in spark width at constant rise times are evidence of a multichannel origin of sparks. Because ridge and ember were reduced by promoters of Ca(2+)-dependent activation (caffeine, low [Mg(2+)]) and became more visible in the presence of its inhibitors, they are probably manifestations of Ca(2+) release directly operated by voltage sensors.

Spatially segregated control of Ca2+ release in developing skeletal muscle of mice.

1. Confocal laser scanning microscopy was used to monitor Ca2+ signals in primary-cultured myotubes, prepared from forelimbs of wild-type or ryanodine receptor type 3 (RyR3) knockout mice. Myotubes loaded with the acetoxymethyl ester (AM) form of fluo-3 were imaged at rest or under whole-cell patch clamp. 2. Discrete Ca2+ release events were detected in intact wild-type and RyR3-knockout myotubes. They showed almost no difference in amplitude and width, but were substantially different in duration. In wild-type myotubes (660 events, 57 cells) the amplitude was 1.27 (0.85, 1.97) (median (25 %, 75 %)) units of resting fluorescence, the full width at half-magnitude (FWHM) was 1.4 (0.9, 2.3) microm, and the full duration at half-magnitude (FDHM) was 25.3 (9.6, 51.7) ms. In RyR3-knockout myotubes (655 events, 83 cells) the amplitude was 1.30 (0.84, 2.08), FWHM was 1.63 (1.02, 2.66) microm, and FDHM was 43.6 (23.6, 76.9) ms. 3. Depolarization under voltage clamp of both wild-type and RyR3-knockout myotubes produced substantial Ca2+ release devoid of discrete Ca2+ events. Discrete events were still present but occurred without correlation with the applied pulse, largely at locations where the pulse did not elicit release. 4. The local correspondence between voltage control and absence of discrete events implies that the functional interaction with voltage sensors suppresses the mechanism that activates discrete events. Because it applies whether RyR3 is present or not, it is this exclusion by voltage of other control mechanisms, rather than isoform composition, that primarily determines the absence of discrete Ca2+ events in adult mammalian muscle.

Calcium release flux underlying Ca2+ sparks of frog skeletal muscle.

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 micrometers. 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 micrometer, 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.

Amplitude distribution of calcium sparks in confocal images: theory and studies with an automatic detection method.

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.

Local calcium release in mammalian skeletal muscle.

1. 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. 2. 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. 3. 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). 4. 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.