Store-operated Ca2+ Entry in Malignant Hyperthermia-susceptible Human Skeletal Muscle

In malignant hyperthermia (MH), mutations in RyR1 underlie direct activation of the channel by volatile anesthetics, leading to muscle contracture and a life-threatening increase in core body temperature. The aim of the present study was to establish whether the associated depletion of sarcoplasmic reticulum (SR) Ca²⁺ triggers sarcolemmal Ca²⁺ influx via store-operated Ca²⁺ entry (SOCE). Samples of vastus medialis muscle were obtained from patients undergoing assessment for MH susceptibility using the in vitro contracture test. Single fibers were mechanically skinned, and confocal microscopy was used to detect changes in [Ca²⁺] either within the resealed t-system ([Ca²⁺]_t-sys) or within the cytosol. In normal fibers, halothane (0.5 mm) failed to initiate SR Ca²⁺ release or Ca²⁺_t-sys depletion. However, in MH-susceptible (MHS) fibers, halothane induced both SR Ca²⁺ release and Ca²⁺_t-sys depletion, consistent with SOCE. In some MHS fibers, halothane-induced SR Ca²⁺ release took the form of a propagated wave, which was temporally coupled to a wave of Ca²⁺_t-sys depletion. SOCE was potently inhibited by “extracellular” application of a STIM1 antibody trapped within the t-system but not when the antibody was denatured by heating. In conclusion, (i) in human MHS muscle, SR Ca²⁺ depletion induced by a level of volatile anesthetic within the clinical range is sufficient to induce SOCE, which is tightly coupled to SR Ca²⁺ release; (ii) sarcolemmal STIM1 has an important role in regulating SOCE; and (iii) sustained SOCE from an effectively infinite extracellular Ca²⁺ pool may contribute to the maintained rise in cytosolic [Ca²⁺] that underlies MH.     

Measurement of sarcoplasmic reticulum Ca2+ content in intact amphibian skeletal muscle fibres with 4-chloro-m-cresol.

Single skeletal muscle fibres were isolated from the toad (Bufo marinus) and isometric force and myoplasmic free calcium concentration ([Ca2+]i) were measured. Brief applications of 4-chloro- m-cresol (4-CmC, 0.2-5 mM) elevated [Ca2+]i reversibly in a dose-dependent manner. The lowest concentration of 4-CmC which reliably gave maximal [Ca2+]i was 2 mM and it was, therefore, used for measurement of sarcoplasmic reticulum (SR) Ca2+ content. Tetanic stimulations (100 Hz) increased [Ca2+]i from a resting level of 105 +/- 47 nM (n = 10) to 1370 +/- 220 nM (n = 6). Application of 2 mM 4-CmC produced a contracture that was 54 +/- 16% (n = 6) of the tetanic force and elevated [Ca2+]i to a peak of 3520 +/- 540 nM (n = 8). Both force and [Ca2+]i levels (resting and tetanic) were restored after 10 min of washout of 4-CmC. In skinned muscle fibres, the myofibrillar Ca(2+)-sensitivity was not changed by 4-CmC, but maximal force was reduced to 74 +/- 10% (n = 4). The magnitude of the peak of the 4-CmC-induced Ca2+ transient was not significantly changed by removal of extracellular Ca2+ nor by inhibiting the SR Ca2+ pump with 2,5-di-tert-butylhydroquinone. Treatment of intact fibres with 30 mM caffeine produced a peak Ca2+ level that was indistinguishable from 2 mM 4-CmC. These results indicate that it is possible to measure the SR Ca2+ content in the same fibre with 4-CmC without loss of normal muscle function.     

Paradoxical buffering of calcium by calsequestrin demonstrated for the calcium store of skeletal muscle

Contractile activation in striated muscles requires a Ca²⁺ reservoir of large capacity inside the sarcoplasmic reticulum (SR), presumably the protein calsequestrin. The buffering power of calsequestrin in vitro has a paradoxical dependence on [Ca²⁺] that should be valuable for function. Here, we demonstrate that this dependence is present in living cells. Ca²⁺ signals elicited by membrane depolarization under voltage clamp were compared in single skeletal fibers of wild-type (WT) and double (d) Casq-null mice, which lack both calsequestrin isoforms. In nulls, Ca²⁺ release started normally, but the store depleted much more rapidly than in the WT. This deficit was reflected in the evolution of SR evacuability, E, which is directly proportional to SR Ca²⁺ permeability and inversely to its Ca²⁺ buffering power, B. In WT mice E starts low and increases progressively as the SR is depleted. In dCasq-nulls, E started high and decreased upon Ca²⁺ depletion. An elevated E in nulls is consistent with the decrease in B expected upon deletion of calsequestrin. The different value and time course of E in cells without calsequestrin indicate that the normal evolution of E reflects loss of B upon SR Ca²⁺ depletion. Decrement of B upon SR depletion was supported further. When SR calcium was reduced by exposure to low extracellular [Ca²⁺], release kinetics in the WT became similar to that in the dCasq-null. E became much higher, similar to that of null cells. These results indicate that calsequestrin not only stores Ca²⁺, but also varies its affinity in ways that progressively increase the ability of the store to deliver Ca²⁺ as it becomes depleted, a novel feedback mechanism of potentially valuable functional implications. The study revealed a surprisingly modest loss of Ca²⁺ storage capacity in null cells, which may reflect concurrent changes, rather than detract from the physiological importance of calsequestrin.     

Increased CaVβ1a expression with aging contributes to skeletal muscle weakness

Ca²⁺ release from the sarcoplasmic reticulum (SR) into the cytosol is a crucial part of excitation–contraction (E‐C) coupling. Excitation–contraction uncoupling, a deficit in Ca²⁺ release from the SR, is thought to be responsible for at least some of the loss in specific force observed in aging skeletal muscle. Excitation–contraction uncoupling may be caused by alterations in expression of the voltage‐dependent calcium channel α_1s (Ca_V1.1) and β_1a (Ca_Vβ_1a) subunits, both of which are necessary for E‐C coupling to occur. While previous studies have found Ca_V1.1 expression declines in old rodents, Ca_Vβ_1a expression has not been previously examined in aging models. Western blot analysis shows a substantial increase of Ca_Vβ_1a expression over the full lifespan of Friend Virus B (FVB) mice. To examine the specific effects of Ca_Vβ_1a overexpression, a Ca_Vβ_1a‐YFP plasmid was electroporated in vivo into young animals. The resulting increase in expression of Ca_Vβ_1a corresponded to decline of Ca_V1.1 over the same time period. YFP fluorescence, used as a measure of Ca_Vβ_1a‐YFP expression in individual fibers, also showed an inverse relationship with charge movement, measured using the whole‐cell patch‐clamp technique. Specific force was significantly reduced in young Ca_Vβ_1a‐YFP electroporated muscle fibers compared with sham‐electroporated, age‐matched controls. siRNA interference of Ca_Vβ_1a in young muscles reduced charge movement, while charge movement in old was restored to young control levels. These studies imply Ca_Vβ_1a serves as both a positive and negative regulator Ca_V1.1 expression, and that endogenous overexpression of Ca_Vβ_1a during old age may play a role in the loss of specific force.     

Chloride-induced Ca2+ Release from the Sarcoplasmic Reticulum of Chemically Skinned Rabbit Psoas Fibers and Isolated Vesicles of Terminal Cisternae

There is increasing evidence that Ca²⁺ release from sarcoplasmic reticulum (SR) of mammalian skeletal muscle is regulated or modified by several factors including ionic composition of the myoplasm. We have studied the effect of Cl⁻ on the release of Ca²⁺ from the SR of rabbit skeletal muscle in both skinned psoas fibers and in isolated terminal cisternae vesicles. Ca²⁺ release from the SR in skinned fibers was inferred from increases in isometric tension and the amount of release was assessed by integrating the area under each tension transient. Ca²⁺ release from isolated SR was measured by rapid filtration of vesicles passively loaded with ⁴⁵Ca²⁺. Ca²⁺ release from SR was stimulated in both preparations by exposure to a solution containing 191 mm choline-Cl, following pre-equilibration in Ca²⁺-loading solution that had propionate as the major anion. Controls using saponin (50 μg/ml), indicated that the release of Ca²⁺ was due to direct action of Cl⁻ on the SR rather than via depolarization of T-tubules. Procaine (10 mm) totally blocked Cl⁻- and caffeine-elicited tension transients recorded using loading and release solutions having ([Na⁺] + [K⁺]) × [Cl⁻] product of 6487.69 mm ² and 12361.52 mm ², respectively, and blocked 60% of Ca²⁺ release in isolated SR vesicles. Surprisingly, procaine had only a minor effect on tension transients elicited by Cl⁻ and caffeine together. The data from both preparations suggests that Cl⁻ induces a relatively small amount of Ca²⁺ release from the SR by activating receptors other than RYR-1. In addition, Cl⁻ may increase the Ca²⁺ sensitivity of RYR-1, which would then allow the small initial release of Ca²⁺ to facilitate further release of Ca²⁺ from the SR by Ca²⁺-induced Ca²⁺ release. Received: 6 February 1996/Revised: 17 July 1996     

Mitochondrial Ca2+-Handling in Fast Skeletal Muscle Fibers from Wild Type and Calsequestrin-Null Mice

Mitochondrial calcium handling and its relation with calcium released from sarcoplasmic reticulum (SR) in muscle tissue are subject of lively debate. In this study we aimed to clarify how the SR determines mitochondrial calcium handling using dCASQ-null mice which lack both isoforms of the major Ca²⁺-binding protein inside SR, calsequestrin. Mitochondrial free Ca²⁺-concentration ([Ca²⁺]_mito) was determined by means of a genetically targeted ratiometric FRET-based probe. Electron microscopy revealed a highly significant increase in intermyofibrillar mitochondria (+55%) and augmented coupling (+12%) between Ca²⁺ release units of the SR and mitochondria in dCASQ-null vs. WT fibers. Significant differences in the baseline [Ca²⁺]_mito were observed between quiescent WT and dCASQ-null fibers, but not in the resting cytosolic Ca²⁺ concentration. The rise in [Ca²⁺]_mito during electrical stimulation occurred in 20−30 ms, while the decline during and after stimulation was governed by 4 rate constants of approximately 40, 1.6, 0.2 and 0.03 s⁻¹. Accordingly, frequency-dependent increase in [Ca²⁺]_mito occurred during sustained contractions. In dCASQ-null fibers the increases in [Ca²⁺]_mito were less pronounced than in WT fibers and even lower when extracellular calcium was removed. The amplitude and duration of [Ca²⁺]_mito transients were increased by inhibition of mitochondrial Na⁺/Ca²⁺ exchanger (mNCX). These results provide direct evidence for fast Ca²⁺ accumulation inside the mitochondria, involvement of the mNCX in mitochondrial Ca²⁺-handling and a dependence of mitochondrial Ca²⁺-handling on intracellular (SR) and external Ca²⁺ stores in fast skeletal muscle fibers. dCASQ-null mice represent a model for malignant hyperthermia. The differences in structure and in mitochondrial function observed relative to WT may represent compensatory mechanisms for the disease-related reduction of calcium storage capacity of the SR and/or SR Ca²⁺-leakage.     

Ca2+ Release in Muscle Fibers Expressing R4892W and G4896V Type 1 Ryanodine Receptor Disease Mutants

The large and rapidly increasing number of potentially pathological mutants in the type 1 ryanodine receptor (RyR1) prompts the need to characterize their effects on voltage-activated sarcoplasmic reticulum (SR) Ca²⁺ release in skeletal muscle. Here we evaluated the function of the R4892W and G4896V RyR1 mutants, both associated with central core disease (CCD) in humans, in myotubes and in adult muscle fibers. For both mutants expressed in RyR1-null (dyspedic) myotubes, voltage-gated Ca²⁺ release was absent following homotypic expression and only partially restored following heterotypic expression with wild-type (WT) RyR1. In muscle fibers from adult WT mice, both mutants were expressed in restricted regions of the fibers with a pattern consistent with triadic localization. Voltage-clamp-activated confocal Ca²⁺ signals showed that fiber regions endowed with G4896V-RyR1s exhibited an ∼30% reduction in the peak rate of SR Ca²⁺ release, with no significant change in SR Ca²⁺ content. Immunostaining revealed no associated change in the expression of either α1S subunit (Cav1.1) of the dihydropyridine receptor (DHPR) or type 1 sarco(endo)plasmic reticulum Ca²⁺ ATPase (SERCA1), indicating that the reduced Ca²⁺ release resulted from defective RyR1 function. Interestingly, in spite of robust localized junctional expression, the R4892W mutant did not affect SR Ca²⁺ release in adult muscle fibers, consistent with a low functional penetrance of this particular CCD-associated mutant.     

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca²⁺ signaling is a fundamental physiological process in living cells. Ca²⁺ sparks are the elementary units of Ca²⁺ signaling in the striated muscle fibers that appear as highly localized Ca²⁺ release events mediated by ryanodine receptor (RyR) Ca²⁺ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca²⁺ sparks could provide information on the intracellular Ca²⁺ handling properties of healthy and diseased striated muscles. Although Ca²⁺ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.Detailed here is an experimental protocol for measuring Ca²⁺ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca²⁺ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca²⁺ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca²⁺ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP₃R) and RyR contributes to Ca²⁺ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca²⁺ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).     

Expression of calcium-binding protein regucalcin mRNA in the cloned human hepatoma cells (HegG2): Stimulation by insulin

We have isolated an endogenous positive inotropic factor (EPIF) from porcine left heart ventricular tissue, which demonstrated to have only weak digitalis-like properties including the inhibition of myocardial Na⁺,K⁺-ATPase. EPIF completely lacks digitalis-like toxicity such as after-contractions in larger doses. In our recent studies, we have demonstrated that EPIF produces a decrease in the amplitude of the post-rest rapid cooling contracture which indicated that EPIF may release Ca²⁺from the sarcoplasmic reticulum. In the present study, the effects of EPIF were investigated on the Ca²⁺uptake and release properties of SR enriched membrane vesicles from rat heart. At pH 6.8 and in the presence of oxalate, EPIF dose-dependently inhibited the ATPdependent uptake of Ca²⁺by SR vesicles. Concentrations as low as 25 ul (in 1 mL uptake medium) of EPIF caused a 45-47% reduction in the uptake of Ca²⁺within 3-4 min. Increases in EPIF concentration to 50 ul/mL caused additional reduction of only 15-20% in the uptake of Ca²⁺. Concentrations of 25 ul/mL of EPIF had little or no effects on passive release of actively loaded Ca²⁺in SR vesicles. On doubling the concentrations to 50 ul/mL EPIF, however, enhanced the release of Ca²⁺by 25-28% during 1-2 min. and 44-48% after 4 min of incubation of Ca²⁺loaded vesicles in the release medium. Relatively smaller effects of EPIF on Ca²⁺release implies that EPIF may mainly lower the uptake of Ca²⁺in SR. This reduced uptake of Ca²⁺may be explained by the EPIF-induced inhibition of Ca²⁺pump.     

Structural and functional properties of ryanodine receptor type 3 in zebrafish tail muscle

The ryanodine receptor (RyR)1 isoform of the sarcoplasmic reticulum (SR) Ca²⁺ release channel is an essential component of all skeletal muscle fibers. RyR1s are detectable as “junctional feet” (JF) in the gap between the SR and the plasmalemma or T-tubules, and they are required for excitation–contraction (EC) coupling and differentiation. A second isoform, RyR3, does not sustain EC coupling and differentiation in the absence of RyR1 and is expressed at highly variable levels. Anatomically, RyR3 expression correlates with the presence of parajunctional feet (PJF), which are located on the sides of the SR junctional cisternae in an arrangement found only in fibers expressing RyR3. In frog muscle fibers, the presence of RyR3 and PJF correlates with the occurrence of Ca²⁺ sparks, which are elementary SR Ca²⁺ release events of the EC coupling machinery. Here, we explored the structural and functional roles of RyR3 by injecting zebrafish (Danio rerio) one-cell stage embryos with a morpholino designed to specifically silence RyR3 expression. In zebrafish larvae at 72 h postfertilization, fast-twitch fibers from wild-type (WT) tail muscles had abundant PJF. Silencing resulted in a drop of the PJF/JF ratio, from 0.79 in WT fibers to 0.03 in the morphants. The frequency with which Ca²⁺ sparks were detected dropped correspondingly, from 0.083 to 0.001 sarcomere⁻¹ s⁻¹. The few Ca²⁺ sparks detected in morphant fibers were smaller in amplitude, duration, and spatial extent compared with those in WT fibers. Despite the almost complete disappearance of PJF and Ca²⁺ sparks in morphant fibers, these fibers looked structurally normal and the swimming behavior of the larvae was not affected. This paper provides important evidence that RyR3 is the main constituent of the PJF and is the main contributor to the SR Ca²⁺ flux underlying Ca²⁺ sparks detected in fully differentiated frog and fish fibers.     

Muscle fibers from senescent mice retain excitation–contraction coupling properties in culture

In the present study, we test the hypothesis that mouse skeletal muscle in culture retains the fundamental properties of excitation-sarcoplasmic reticulum Ca²⁺ release coupling reported for young-adult (3–4 mo) and senescent (22–23) mice. Dissociated flexor digitorum brevis (FDB) muscles from young-adult and senescent mice were cultured for 7 d in a serum-free medium. During this period, the overall morphology of cultured fibers resembled that exhibited by acutely dissociated cells. In addition, survival analysis revealed that more than 70% of the fibers from both young and old mice remained suitable for electrophysiological studies during this same culture period. Charge movement and intracellular Ca²⁺ recordings in FDB fibers, voltage clamped in the whole cell configuration of the patch-clamp technique, reproduced the maximal values, and voltage dependence similarly displayed by acutely dissociated cells for both parameters in young-adult and senescent mice. The analysis of the dihydropyridine receptor by immunoblots confirmed, in the culture system, the age-dependent decrease in the expression of this protein. In conclusion, FDB fibers from young-adult and old mice retain the excitation–contraction coupling phenotype during the course of a week in serum-free medium culture.     

Functional Effects of a Restrictive-Cardiomyopathy-Linked Cardiac Troponin I Mutation (R145W) in Transgenic Mice

The human cardiac troponin I (hcTnI) mutation R145W has been associated with restrictive cardiomyopathy. In this study, simultaneous measurements of ATPase activity and force in skinned papillary fibers from hcTnI R145W transgenic mice (Tg-R145W) were explored. Tg-R145W fibers showed an ∼ 13-16% increase in maximal Ca²⁺-activated force and ATPase activity compared to hcTnI wild-type transgenic mice. The force-generating cross-bridge turnover rate (g) and the energy cost (ATPase/force) were the same in all groups of fibers. Also, the Tg-R145W fibers showed a large increase in the Ca²⁺ sensitivity of both force development and ATPase. In intact fibers, the mutation caused prolonged force and intracellular [Ca²⁺] transients and increased time to peak force. Analysis of force and Ca²⁺ transients showed that there was a 40% increase in peak force in Tg-R145W muscles, which was likely due to the increased Ca²⁺ transient duration. The above cited results suggest that: (1) there would be an increase in resistance to ventricular filling during diastole resulting from the prolonged force and Ca²⁺ transients that would result in a decrease in ventricular filling (diastolic dysfunction); and (2) there would be a large (approximately 53%) increase in force during systole, which may help to partly compensate for diastolic dysfunction. These functional results help to explain the mechanisms by which these mutations give rise to a restrictive phenotype.     

Predicting Local SR Ca Dynamics during Ca Wave Propagation in Ventricular Myocytes

Of the many ongoing controversies regarding the workings of the sarcoplasmic reticulum (SR) in cardiac myocytes, two unresolved and interconnected topics are 1), mechanisms of calcium (Ca²⁺) wave propagation, and 2), speed of Ca²⁺ diffusion within the SR. Ca²⁺ waves are initiated when a spontaneous local SR Ca²⁺ release event triggers additional release from neighboring clusters of SR release channels (ryanodine receptors (RyRs)). A lack of consensus regarding the effective Ca²⁺ diffusion constant in the SR (D_Ca,SR) severely complicates our understanding of whether dynamic local changes in SR [Ca²⁺] can influence wave propagation. To address this problem, we have implemented a computational model of cytosolic and SR [Ca²⁺] during Ca²⁺ waves. Simulations have investigated how dynamic local changes in SR [Ca²⁺] are influenced by 1), D_Ca,SR; 2), the distance between RyR clusters; 3), partial inhibition or stimulation of SR Ca²⁺ pumps; 4), SR Ca²⁺ pump dependence on cytosolic [Ca²⁺]; and 5), the rate of transfer between network and junctional SR. Of these factors, D_Ca,SR is the primary determinant of how release from one RyR cluster alters SR [Ca²⁺] in nearby regions. Specifically, our results show that local increases in SR [Ca²⁺] ahead of the wave can potentially facilitate Ca²⁺ wave propagation, but only if SR diffusion is relatively slow. These simulations help to delineate what changes in [Ca²⁺] are possible during SR Ca²⁺release, and they broaden our understanding of the regulatory role played by dynamic changes in [Ca²⁺]_SR.     

Ca2+-Activated Force Production and Calcium Handling by the Sarcoplasmic Reticulum of Crustacean Muscles During Molt-Induced Atrophy

Growth in crustaceans is an intermittent process centered aroundthe principal event of ecdysis. A major problem facing decapodcrustaceans at the time of ecdysis is the withdrawal of thelarge muscle mass of the chelae through the narrow basi-ischialjoints. To overcome this problem the muscle undergoes an atrophytriggered by the molt, which reduces the muscle mass. Once theanimal is freed from the old exoskeleton, the muscle fibers,must elongate to accommodate the new larger exoskeleton. Despitethis major myofibrillar remodification, the muscles are thoughtto remain functional over the molt cycle. Studies using skinnedmuscle fibers have shown that long-sarcomere fibers maintaintheir function over the molt cycle while the contractile propertiesof the short-sarcomere fibers are modified, as fibers couldnot withstand maximal activation with Ca²⁺ during the premoltstage. In this study the maximum Ca²⁺-activated force productionand the ability of the sarcoplasmic reticulum (SR) to releaseaccumulated Ca²⁺ has been investigated in the two major fibertypes in the claw muscle of Cherax destructor, in the stagesjust prior to ecdysis and during inter molt. In both long- andshort-sarcomere fibers, the amount of Ca²⁺ released by the SRwas not different in premolt and intermolt stages. However,the maximum releasing capacity of the SR was reached in a shortertime during the premolt suggesting that Ca²⁺ is being accumulatedat a faster rate. The force production was greatly reduced andwas graded during the premolt in both fiber types. This modulationof force appears to be the most likely candidate regulatingthe magnitude of the force development in the periods when fibersare undergoing myofibrillar remodification and thus may serveto prevent fiber damage.     

Ca2+ Overload and Sarcoplasmic Reticulum Instability in tric-a Null Skeletal Muscle

The sarcoplasmic reticulum (SR) of skeletal muscle contains K⁺, Cl⁻, and H⁺ channels may facilitate charge neutralization during Ca²⁺ release. Our recent studies have identified trimeric intracellular cation (TRIC) channels on SR as an essential counter-ion permeability pathway associated with rapid Ca²⁺ release from intracellular stores. Skeletal muscle contains TRIC-A and TRIC-B isoforms as predominant and minor components, respectively. Here we test the physiological function of TRIC-A in skeletal muscle. Biochemical assay revealed abundant expression of TRIC-A relative to the skeletal muscle ryanodine receptor with a molar ratio of TRIC-A/ryanodine receptor ∼5:1. Electron microscopy with the tric-a^−/− skeletal muscle showed Ca²⁺ overload inside the SR with frequent formation of Ca²⁺ deposits compared with the wild type muscle. This elevated SR Ca²⁺ pool in the tric-a^−/− muscle could be released by caffeine, whereas the elemental Ca²⁺ release events, e.g. osmotic stress-induced Ca²⁺ spark activities, were significantly reduced likely reflecting compromised counter-ion movement across the SR. Ex vivo physiological test identified the appearance of “alternan” behavior with isolated tric-a^−/− skeletal muscle, i.e. transient and drastic increase in contractile force appeared within the decreasing force profile during repetitive fatigue stimulation. Inhibition of SR/endoplasmic reticulum Ca^{2+ ATPase} function could lead to aggravation of the stress-induced alternans in the tric-a^−/− muscle. Our data suggests that absence of TRIC-A may lead to Ca²⁺ overload in SR, which in combination with the reduced counter-ion movement may lead to instability of Ca²⁺ movement across the SR membrane. The observed alternan behavior with the tric-a^−/− muscle may reflect a skeletal muscle version of store overload-induced Ca²⁺ release that has been reported in the cardiac muscle under stress conditions.     

Increased calcium release from sarcoplasmic reticulum stimulated by inositol trisphosphate in spontaneously hypertensive rat heart cells

It is known that inositol (1, 4, 5)-trisphosphate (IP₃) stimulates Ca²⁺ release from sarcoplasmic reticulum (SR) in several tissues, but in cardiac myocytes this phenomenon has not been confirmed. The purpose of the present study was to confirm the effect of (1, 4, 5)-IP₃ on Ca²⁺ release from SR in cardiac myocytes. The effect of IP₃ on Ca²⁺ release from SR in hypertrophic cardiac cells was also determined.We examined the effects of IP₃ on Ca²⁺ release from cardiac myocyte SR by the bigital-image method in a single cell. We also determined the effect of IP₃ on calcium release from isolated SR. SR was prepared from spontaneous hypertensive rat hearts and Wistar kyoto rat hearts. The SR was prelabeled with⁴⁵Ca²+, and then incubated with the indicated concentrations of IP³ for 1 min at 37°C. In cardiac myocytes treated with saponin, Ca²⁺ release stimulated by 10 M (1, 4, 5)-IP₃ was detected by fura-2. In⁴⁵Ca²⁺ prelabeled SR, the maximal Ca²⁺ release was achieved at 10 M IP₃ incubated for 1 min. The release of Ca²⁺ was higher in Sr of SHR than in the SR of WKY. IP₃ stimulates Ca²⁺ release from cardiac SR, and this release is greater in SHR than in WKY. However, it is uncertain whether this phenomenon plays a role in cardiac hypertrophy.     

Effect of calmodulin on sarcoplasmic reticular Ca2+-transport in the aging heart

Heart failure is common among the elderly and an alteration in myocardial Ca²⁺ transport is believed to be involved in its depressed contractile performance. Although ATP-dependent sarcoplasmic reticular (SR) Ca²⁺ transport has been reported to decrease in old hearts, virtually nothing appears to be known about the Ca²⁺ pump activity of SR in aging myocardium in the presence of calmodulin, one of its endogenous activators. In this study, the activity of the Ca²⁺ pump of aging cardiac SR was assessed in the presence of this endogenous stimulator. This assessment was therefore designed to give additional information about the status of this enzyme in old hearts. Male Sprague-Dawley rats were used and were divided into 3 groups: young (4–6 months old); middle-aged (15–17 months old) and old age (24–25 months old). Purified SR membranes were isolated from ventricular tissues. ATP-dependent Ca²⁺ accumulation by membrane vesicles of middle-aged and old hearts was significantly depressed in comparison to young hearts at all Ca²⁺ concentrations employed in the absence and presence of calmodulin. The activity of this Ca²⁺ transporter was similar in middle-aged and old hearts even in the presence of calmodulin. These results suggest that the activity of the Ca²⁺ pump in SR of aging hearts is depressed even in the presence of calmodulin.C. E. Heyliger is a Scholar of the British Columbia Heart Foundation.     

Control of Ca Release by Action Potential Configuration in Normal and Failing Murine Cardiomyocytes

Cardiomyocytes from failing hearts exhibit spatially nonuniform or dyssynchronous sarcoplasmic reticulum (SR) Ca²⁺ release. We investigated the contribution of action potential (AP) prolongation in mice with congestive heart failure (CHF) after myocardial infarction. AP recordings from CHF and control myocytes were included in a computational model of the dyad, which predicted more dyssynchronous ryanodine receptor opening during stimulation with the CHF AP. This prediction was confirmed in cardiomyocyte experiments, when cells were alternately stimulated by control and CHF AP voltage-clamp waveforms. However, when a train of like APs was used as the voltage stimulus, the control and CHF AP produced a similar Ca²⁺ release pattern. In this steady-state condition, greater integrated Ca²⁺ entry during the CHF AP lead to increased SR Ca²⁺ content. A resulting increase in ryanodine receptor sensitivity synchronized SR Ca²⁺ release in the mathematical model, thus offsetting the desynchronizing effects of reduced driving force for Ca²⁺ entry. A modest nondyssynchronous prolongation of Ca²⁺ release was nevertheless observed during the steady-state CHF AP, which contributed to increased time-to-peak measurements for Ca²⁺ transients in failing cells. Thus, dyssynchronous Ca²⁺ release in failing mouse myocytes does not result from electrical remodeling, but rather other alterations such as T-tubule reorganization.     

Regulation of sarcoplasmic reticulum Ca2+ release by cytosolic glutathione in rabbit ventricular myocytes

Of the major cellular antioxidant defenses, glutathione (GSH) is particularly important in maintaining the cytosolic redox potential. Whereas the healthy myocardium is maintained at a highly reduced redox state, it has been proposed that oxidation of GSH can affect the dynamics of Ca²⁺-induced Ca²⁺ release. In this study, we used multiple approaches to define the effects of oxidized glutathione (GSSG) on ryanodine receptor (RyR)-mediated Ca²⁺ release in rabbit ventricular myocytes. To investigate the role of GSSG on sarcoplasmic reticulum (SR) Ca²⁺ release induced by the action potential, we used the thiol-specific oxidant diamide to increase intracellular GSSG in intact myocytes. To more directly assess the effect of GSSG on RyR activity, we introduced GSSG within the cytosol of permeabilized myocytes. RyR-mediated Ca²⁺ release from the SR was significantly enhanced in the presence of GSSG. This resulted in decreased steady-state diastolic [Ca²⁺]_SR, increased SR Ca²⁺ fractional release, and increased spark- and non-spark-mediated SR Ca²⁺ leak. Single-channel recordings from RyR’s incorporated into lipid bilayers revealed that GSSG significantly increased RyR activity. Moreover, oxidation of RyR in the form of intersubunit crosslinking was present in intact myocytes treated with diamide and permeabilized myocytes treated with GSSG. Blocking RyR crosslinking with the alkylating agent N-ethylmaleimide prevented depletion of SR Ca²⁺ load induced by diamide. These findings suggest that elevated cytosolic GSSG enhances SR Ca²⁺ leak due to redox-dependent intersubunit RyR crosslinking. This effect can contribute to abnormal SR Ca²⁺ handling during periods of oxidative stress.     

Modulation of the Frequency of Spontaneous Sarcoplasmic Reticulum Ca2+ Release Events (Ca2+ Sparks) by Myoplasmic [Mg2+] in Frog Skeletal Muscle

The modulation by internal free [Mg²⁺] of spontaneous calcium release events (Ca²⁺ “sparks”) from the sarcoplasmic reticulum (SR) was studied in depolarized notched frog skeletal muscle fibers using a laser scanning confocal microscope in line-scan mode (x vs. t). Over the range of [Mg²⁺] from 0.13 to 1.86 mM, decreasing the [Mg²⁺] induced an increase in the frequency of calcium release events in proportion to [Mg²⁺]^−1.6. The change of event frequency was not due to changes in [Mg-ATP] or [ATP]. Analysis of individual SR calcium release event properties showed that the variation in event frequency induced by the change of [Mg²⁺] was not accompanied by any changes in the spatiotemporal spread (i.e., spatial half width or temporal half duration) of Ca²⁺ sparks. The increase in event frequency also had no effect on the distribution of event amplitudes. Finally, the rise time of calcium sparks was independent of the [Mg²⁺], indicating that the open time of the SR channel or channels underlying spontaneous calcium release events was not altered by [Mg²⁺] over the range tested. These results suggest that in resting skeletal fibers, [Mg²⁺] modulates the SR calcium release channel opening frequency by modifying the average closed time of the channel without altering the open time. A kinetic reaction scheme consistent with our results and those of bilayer and SR vesicle experiments indicates that physiological levels of resting Mg²⁺ may inhibit channel opening by occupying the site for calcium activation of the SR calcium release channel.