Myoplasmic Calcium Concentration Research Articles

X-ROS Signaling Depends on Length-Dependent Calcium Buffering by Troponin.

The stretching of a cardiomyocyte leads to the increased production of reactive oxygen species that increases ryanodine receptor open probability through a process termed X-ROS signaling. The stretching of the myocyte also increases the calcium affinity of myofilament Troponin C, which increases its calcium buffering capacity. Here, an integrative experimental and modeling study is pursued to explain the interplay of length-dependent changes in calcium buffering by troponin and stretch-activated X-ROS calcium signaling. Using this combination, we show that the troponin C-dependent increase in myoplasmic calcium buffering during myocyte stretching largely offsets the X-ROS-dependent increase in calcium release from the sarcoplasmic reticulum. The combination of modeling and experiment are further informed by the elimination of length-dependent changes to troponin C calcium binding in the presence of blebbistatin. Here, the model suggests that it is the X-ROS signaling-dependent Ca2+ release increase that serves to maintain free myoplasmic calcium concentrations during a change in myocyte length. Together, our experimental and modeling approaches have further defined the relative contributions of X-ROS signaling and the length-dependent calcium buffering by troponin in shaping the myoplasmic calcium transient.

BMI and malignant hyperthermia pathogenic ryanodine receptor type 1 sequence variants in Switzerland: A retrospective cohort analysis.

Ryanodine receptor type 1 (RYR1) sequence variants are pathogenic for malignant hyperthermia. Variant carriers have a subtle increase in resting myoplasmic calcium concentration compared with nonaffected individuals, but whether this has metabolic effects in daily life is unknown. We analysed the potential effect of malignant hyperthermia-pathogenic RYR1 sequence variants on BMI as a single factor. Due to the heterogeneity of genetic variants predisposing to malignant hyperthermia, and to incomplete information about their regional distribution, we describe the prevalence of RYR1 variants in our population. A retrospective cohort study. A single University hospital. Patients from malignant hyperthermia families with pathogenic RYR1 sequence variants were selected if BMI was available. BMI values were compared amongst malignant hyperthermia susceptible (MHS) and malignant hyperthermia-negative individuals using hierarchical multivariable analyses adjusted for age and sex and considering family clustering. Variant prevalence was calculated. The study included 281 individuals from 42 unrelated malignant hyperthermia families, 109 of whom were MHS and carriers of the familial RYR1 sequence variants. Median [IQR] BMI in MHS individuals with pathogenic RYR1 variants was 22.5 kg m-2 [21.3 to 25.6 kg m-2]. In malignant hyperthermia-negative individuals without variants, median BMI was 23.4 kg m-2 [21.0 to 26.3 kg m-2]. Using multivariable regression adjusted for age and sex, the mean difference was -0.73 (95% CI -1.51 to 0.05). No carrier of a pathogenic RYR1 sequence variant was found to have BMI higher than 30 kg m-2. Only 10 RYR1 variants from the list of the European MH Group were found in our cohort, the most common being p.Val2168Met (39% of families), p.Arg2336His (24%) and p.Arg614Cys (12%). The observed tendency towards lower BMI values in carriers of malignant hyperthermia-pathogenic RYR1 sequence variants points to a possible protective effect on obesity. This study confirms regional differences of the prevalence of malignant hyperthermia-pathogenic RYR1 sequence variants, with just three variants covering 75% of Swiss MHS families. This manuscript is based on a retrospective analysis.

Role of Ca2+ in changing active force during intermittent submaximal stimulation in intact, single mouse muscle fibers

Fatigue of single mouse fibers during repeated high-frequency stimulation results initially from decreased Ca2+ sensitivity while free myoplasmic calcium concentration ([Ca2+]m) increases, followed by decreasing [Ca2+]m. Recovery of active force with low-frequency stimulation is slow and persistent fatigue results from low [Ca2+]m. However, the consequences of intermittent submaximal contractions are not known. The aim of the present study was to investigate the changes in [Ca2+]m and active force during intermittent submaximal contractions and subsequent recovery. Single fibers of mouse flexor digitorum brevis muscles at 32 °C were stimulated with 40 or 50 Hz, for 350 ms every 2 s for 2 min and then every 1 s until < 40% of initial force. Values obtained during the intermittent stimulation were compared with a control force-[Ca2+]m relationship. A “P”-shaped pattern in the force-[Ca2+]m relationship was observed during intermittent stimulation. Early in the intermittent stimulation, [Ca2+]m increased while active force decreased. Subsequent force potentiation was accompanied by increased Ca2+ sensitivity. Later, as active force declined, [Ca2+]m decreased significantly (p < 0.001). This was followed, in the final phase, by a significant decrease in Ca2+ sensitivity determined by [Ca2+]m at half-maximal force (Ca50) (p = 0.001). Low-frequency fatigue persisted during recovery while Ca50 was not significantly different from prefatigue (p > 0.5). In conclusion, the main mechanism of fatigue is due to decreases in both [Ca2+]m and Ca2+ sensitivity following the initial force potentiation. The intermittent submaximal contractions resulted in persistent low-frequency fatigue seen during recovery, which was explained by depressed [Ca2+]m with no change in Ca2+ sensitivity.

Non-crossbridge stiffness in active muscle fibres.

Stretching of an activated skeletal muscle induces a transient tension increase followed by a period during which the tension remains elevated well above the isometric level at an almost constant value. This excess of tension in response to stretching has been called 'static tension' and attributed to an increase in fibre stiffness above the resting value, named 'static stiffness'. This observation was originally made, by our group, in frog intact muscle fibres and has been confirmed more recently, by us, in mammalian intact fibres. Following stimulation, fibre stiffness starts to increase during the latent period well before crossbridge force generation and it is present throughout the whole contraction in both single twitches and tetani. Static stiffness is dependent on sarcomere length in a different way from crossbridge force and is independent of stretching amplitude and velocity. Static stiffness follows a time course which is distinct from that of active force and very similar to the myoplasmic calcium concentration time course. We therefore hypothesize that static stiffness is due to a calcium-dependent stiffening of a non-crossbridge sarcomere structure, such as the titin filament. According to this hypothesis, titin, in addition to its well-recognized role in determining the muscle passive tension, could have a role during muscle activity.

Modeling Local X-ROS and Calcium Signaling in the Heart

Stretching single ventricular cardiac myocytes has been shown experimentally to activate transmembrane nicotinamide adenine dinucleotide phosphate oxidase type 2 to produce reactive oxygen species (ROS) and increase the Ca2+ spark rate in a process called X-ROS signaling. The increase in Ca2+ spark rate is thought to be due to an increase in ryanodine receptor type 2 (RyR2) open probability by direct oxidation of the RyR2 protein complex. In this article, a computational model is used to examine the regulation of ROS and calcium homeostasis by local, subcellular X-ROS signaling and its role in cardiac excitation-contraction coupling. To this end, a four-state RyR2 model was developed that includes an X-ROS-dependent RyR2 mode switch. When activated, [Ca2+]i-sensitive RyR2 open probability increases, and the Ca2+ spark rate changes in a manner consistent with experimental observations. This, to our knowledge, new model is used to study the transient effects of diastolic stretching and subsequent ROS production on RyR2 open probability, Ca2+ sparks, and the myoplasmic calcium concentration ([Ca2+]i) during excitation-contraction coupling. The model yields several predictions: 1) [ROS] is produced locally near the RyR2 complex during X-ROS signaling and increases by an order of magnitude more than the global ROS signal during myocyte stretching; 2) X-ROS activation just before the action potential, corresponding to ventricular filling during diastole, increases the magnitude of the Ca2+ transient; 3) during prolonged stretching, the X-ROS-induced increase in Ca2+ spark rate is transient, so that long-sustained stretching does not significantly increase sarcoplasmic reticulum Ca2+ leak; and 4) when the chemical reducing capacity of the cell is decreased, activation of X-ROS signaling increases sarcoplasmic reticulum Ca2+ leak and contributes to global oxidative stress, thereby increases the possibility of arrhythmia. The model provides quantitative information not currently obtainable through experimental means and thus provides a framework for future X-ROS signaling experiments.

Heat Production and the Importance of Temperature Management in Malignant Hyperthermia

Malignant hyperthermia (MH) is a rare pharmacogenetic, subclinical disorder of the striated skeletal muscle. Acute episodes are usually triggered by exposure to halogenated volatile anesthetics and/or succinylcholine. During an MH episode, the patient's muscles go into a hypermetabolic state, which in turn causes variable clinical signs and symptoms: i.e., muscle rigidity, respiratory and metabolic acidosis, muscle breakdown, cardiac responses (tachycardia) and ultimately elevation of body temperature. At the cellular level, MH is the consequence of abnormally sustained high myoplasmic calcium concentrations, which causes dramatically elevated turnover of energy rich compounds (e.g., adenosine triphosphate (ATP)). Although often a late sign of MH, the production of heat can play an important role in the treatment and clinical consequences of an elicited episode. In other words, the production of heat thereby is not merely a simple consequence of MH, but a substrate of high relevance during an ongoing MH event: it has been suggested that heat itself may trigger MH in some genetic mutations. Since the introduction of dantrolene, which has provided a specific MH therapy by normalizing myoplasmic calcium levels, mortality of MH has decreased from 70% to 4%. Nevertheless, strong consideration for proper temperature management of the known or suspected MH patient, in all clinical areas (pre-, intra- and post-operative), is critical: as hyperthermia can cause multisystemic involvements, including the brain, the heart and the hemostatic system.

New insights on sarcoplasmic reticulum calcium regulation in muscle fatigue

a consistent observation with fatigue in skeletal muscle is a decline in the amplitude of the myoplasmic Ca2+ transient, which is thought to result primarily from a reduced Ca2+ flux through the ryanodine receptor (RyR1) of the sarcoplasmic reticulum (SR) ([Fig. 1][1]). This in turn is thought to

Verapamil and NAC reduce the excess and damaging increase in myoplasmic calcium concentration during fatigue

Muscles that lack KATP channel activity generate much greater resting [Ca2+]i and force than normal muscles. The large increase in resting force in KATP channel deficient muscles is completely abolished with 1 μM verapamil, a L-type Ca2+ channel blocker, which suggests that the increase in resting force was due to a Ca2+ influx through L-type Ca2+ channel. However, we recently found that NAC, a ROS scavenger, also reduces resting force. The objective of this study was to test the hypothesis that “the excess increases in resting [Ca2+]i during fatigue in KATP channel deficient muscles starts with an excess Ca2+ influx through L-type Ca2+ channels, followed by excess ROS production that somehow causes a further increase in resting [Ca2+]i”. At 1 μm, verapamil had no effect on tetanic and resting Ca2+ before fatigue, but significantly reduced resting [Ca2+]i in KATP channel deficient fibers. At 1 mM, NAC did not affect contractility and reduced the increase in resting [Ca2+]i in KATP channel deficient fibers. It is therefore suggested that the excess increased in resting [Ca2+]i during fatigue in KATP channel deficient FDB fibers is not completely due to an influx through L-type Ca2+ channels as it may involve excess ROS production acting on proteins that regulate [Ca2+]i (e.g., the Ca2+ ATPase).

Propofol-Induced Changes in Myoplasmic Calcium Concentrations in Cultured Human Skeletal Muscles from RYR1 Mutation Carriers

Malignant hyperthermia is a pharmacogenetic disorder caused by autosomal dominant mutations in the ryanodine receptor type 1 gene. Propofol has been reported as a safe anaesthetic for malignant hyperthermia susceptible patients but has not been tested on cultured cells from patients with the ryanodine receptor type 1 mutation. The aim of this study was to determine whether propofol could trigger abnormal calcium fluxes in human myotubes isolated from malignant hyperthermia susceptible patients harbouring the native ryanodine receptor type 1 mutation. Muscle specimens were obtained from the patients to diagnose malignant hyperthermia disposition and the calcium-induced calcium release test and molecular genetic analyses were performed. Using the calcium sensitive probe Fura 2, we determined the 340/380 nm wave-length ratios by measuring alterations in calcium homeostasis in isolated myotubes from cultured skeletal muscle specimens. Two patients, one with ryanodine receptor type 1 R2508C and one with the L4838V mutation had accelerated calcium-induced calcium release rates. The 340/380 nm ratios increased when the propofol concentration exceeded 100 microM. The half-maximal activation concentrations (EC50) for propofol from patients 1 and 2 were 181.1 and 420.5 microM, respectively. Increases in calcium concentrations in response to propofol dosage were limited to doses at least 100-fold greater than those used in clinical settings. These observations correlate well with clinical observations that propofol does not trigger malignant hyperthermia in susceptible humans.

Propofol-induced changes of myoplasmic calcium concentration in cultured human skeletal muscles from RYR1 mutation carriers

Propofol-induced changes of myoplasmic calcium concentration in cultured human skeletal muscles from RYR1 mutation carriers

Myoplasmic calcium regulation in myotubes from horses with recurrent exertional rhabdomyolysis.

To determine whether alterations in myoplasmic calcium regulation can be identified in muscle cell cultures (myotubes) and intact muscle fiber bundles derived from Thoroughbreds affected with recurrent exertional rhabdomyolysis (RER). 6 related Thoroughbreds with RER and 8 clinically normal (control) Thoroughbred or crossbred horses. Myotube cell cultures were grown from satellite cells obtained from muscle biopsy specimens of RER-affected and control horses. Fura-2 fluorescence was used to measure resting myoplasmic calcium concentration as well as caffeine- and 4-chloro-m-cresol (4-CMC)-induced increases in myoplasmic calcium. In addition, intact intercostal muscle fiber bundles were prepared from both types of horses, and their sensitivities to caffeine- and 4-CMC-induced contractures were determined. Myotubes of RER-affected and control horses had identical resting myoplasmic calcium concentrations. Myotubes from RER-affected horses had significantly higher myoplasmic calcium concentrations than myotubes from control horses following the addition of > or = 2mM caffeine; however, there was no difference in their response to 4-CMC (> or = 1 mM). Caffeine contracture thresholds for RER and control intact muscle cell bundles (2 vs 10mM, respectively) were significantly different, but 4-CMC contracture thresholds of muscle bundles from RER-affected and control horses (500 microM) did not differ. An increase in caffeine sensitivity of muscle cells derived from a family of related RER-affected horses was detected in vitro by use of cell culture with calcium imaging and by use of fiber bundle contractility techniques. An alteration in muscle cell calcium regulation is a primary factor in the cause of this heritable myopathy.

Functional P2Y2 nucleotide receptors mediate uridine 5'-triphosphate-induced intimal hyperplasia in collared rabbit carotid arteries.

Extracellular uridine 5'-triphosphate (UTP) induces mitogenic activation of smooth muscle cells (SMCs) through binding to P2Y2 nucleotide receptors. P2Y2 receptor mRNA is upregulated in intimal lesions of rat aorta, but it is unclear how this G-protein-coupled receptor contributes to development of intimal hyperplasia. This study used a silicone collar placed around rabbit carotid arteries to induce vascular injury and intimal thickening. Collar placement caused rapid upregulation of P2Y2 receptor mRNA in medial SMCs before appearance of neointima. Fura-2 digital imaging of single SMCs was used to measure changes in myoplasmic calcium concentration (Ca(m)) in response to P2Y receptor agonists. In contrast to UDP, activation by UTP or adenosine 5'-triphosphate (ATP) greatly increased Ca(m), which indicates upregulation of functional P2Y2 receptors at which UTP and ATP are equipotent agonists. The number of responsive cells was significantly greater for freshly dispersed SMCs from collared arteries than for controls. Perivascular infusion of UTP (100 micromol/L) within the collar significantly enhanced neointimal development. Intimas that resulted from UTP exposure were infiltrated by macrophages. Moreover, increased expression of osteopontin occurred in response to in situ application of UTP. ATP or UTP also stimulated osteopontin expression in cultured SMCs in a dose-dependent manner. Furthermore, P2Y2 antisense oligonucleotide inhibited osteopontin expression induced by UTP. These findings indicate for the first time a role for the UTP/ATP receptor, P2Y2, in development of intimal hyperplasia associated with atherosclerosis and restenosis.

Phenotyping malignant hyperthermia susceptibility by measuring halothane-induced changes in myoplasmic calcium concentration in cultured human skeletal muscle cells

Malignant hyperthermia (MH) is a potentially lethal disease triggered by volatile anaesthetics and succinylcholine in genetically predisposed individuals. Because of the heterogenetic nature of MH, a simple genetic-based diagnostic test is not feasible and diagnosis requires an invasive open muscle biopsy followed by the in vitro contracture test (IVCT). Our aim was to establish if measurements of halothane-induced increases in intracellular calcium ion concentration [Ca(2+)](i) in cultured human skeletal muscle cells can be used to phenotype MH susceptibility and if different mutations in the ryanodine receptor (RYR1) gene affect halothane-induced increases in [Ca(2+)](i). Primary cultures of human skeletal muscle cells were established from 54 individuals diagnosed by the IVCT according to the protocol of the European MH Group as: MH susceptible (n=22), MH negative (n=18) or MH equivocal (n=14). All individuals were screened for the presence of the most common mutations in the RYR1 gene. [Ca(2+)](i) was measured by fluorescent digital microscopy using fura-2/AM in 10 cells from each patient at five different halothane concentrations. The halothane-induced increase in [Ca(2+)](i) differed significantly between the three diagnostic groups. Different mutations of the RYR1 gene did not have a specific impact on halothane-induced increases in [Ca(2+)](i). Measurements of [Ca(2+)](i) in human skeletal muscle cells can be used to phenotype MH susceptibility; however, we did not observe a specific effect of any mutation in the RYR1 gene on the halothane-induced increase in [Ca(2+)](i).

Na+–Ca2+ exchange activity in rat skeletal myotubes: effect of lithium ions

The whole-cell patch-clamp technique coupled with intracellular [Ca2+] measurements was used to investigate the sodium–calcium exchange mechanism in rat skeletal muscle cells in primary culture. Replacing external Na+ ions with Li+ or N-methyl-D-glucamine (NMDG+) ions generated outward currents which were correlated with significant increases of free cytosolic-calcium concentration. These results strongly argue for a functional Na+–Ca2+ exchange mechanism working in its reverse mode. Moreover, the outward currents were sensitive to the new compound KB-R7943 (10 μM), which has been shown to be a potent inhibitor of the sodium–calcium exchanger. Outward Na+–Ca2+ exchange current densities were reduced in the presence of external Li+ as compared to those measured in the presence of NMDG+. After replacing internal sodium by lithium ions, rapid changes of external lithium concentrations generated sarcolemmal currents which were accompanied by subsequent variations of intracellular calcium activity. The currents were dependent on extracellular Li+ with a half-maximal activation at 67 mM and a Hill coefficient of 2.9. This work shows that the Na+–Ca2+ exchanger is able to significantly influence the myoplasmic calcium concentration of cultured rat myotubes. On the other hand, our results suggest that Li+ ions may substitute Na+ ions to catalyse an electrogenic Li+/Ca2+ counter transport.

Calcium sensitivity of force production and myofibrillar ATPase activity in muscles from Thoroughbreds with recurrent exertional rhabdomyolysis.

To determine whether the basis for recurrent exertional rhabdomyolysis (RER) in Thoroughbreds lies in an alteration in the activation and regulation of the myofibrillar contractile apparatus by ionized calcium. 4 Thoroughbred mares with RER and 4 clinically normal (control) Thoroughbreds. Single chemically-skinned type-I (slow-twitch) and type-II (fast-twitch) muscle fibers were obtained from punch biopsy specimens, mounted to a force transducer, and the tensions that developed in response to a series of calcium concentrations were measured. In addition, myofibril preparations were isolated from muscle biopsy specimens and the maximal myofibrillar ATPase activity, as well as its sensitivity to ionized calcium, were measured. Equine type-I muscle fibers were more readily activated by calcium than were type-II muscle fibers. However, there was no difference between the type-II fibers of RER-affected and control horses in terms of calcium sensitivity of force production. There was also no difference between muscle myofibril preparations from RER-affected and control horses in calcium sensitivity of myofibrillar ATPase activity. An alteration in myofibrillar calcium sensitivity is not a basis for pathologic contracture development in muscles from RER-affected horses. Recurrent exertional rhabdomyolysis in Thoroughbreds may represent a novel heritable defect in the regulation of muscle excitation-contraction coupling or myoplasmic calcium concentration.

Effect of carticaine on the sarcoplasmic reticulum Ca2+-dependent adenosine triphosphatase.

The sarcoplasmic reticulum Ca2+-ATPase (calcium-dependent adenosine triphosphatase) transports Ca2+ from the myoplasm to the reticulum lumen at the expense of free energy from ATP hydrolysis. Carticaine is a local anesthetic of frequent use in dentistry which is now entering other clinical fields. We studied the action of carticaine on the sarcoplasmic reticulum (SR) skeletal muscle Ca2+-ATPase. SR vesicles from rabbit fast skeletal muscle were used. Carticaine inhibits the enzymatic activity. The inhibition of the enzymatic activity depends on pH, [Ca2+] and the presence of calcimycin. Half-maximal carticaine concentration that inhibits the ATPase activity tends to a maximal value upon increasing [Ca2+]. Carticaine concentrations required to inhibit the enzymatic activity at myoplasmic calcium concentration are lower than usual clinical doses: Ki=6.0+/-1.4 mM carticaine (n=5) for 0.1 microM [Ca2+]. ATP-dependent calcium uptake is also inhibited by the local anesthetic: Ki=30.5+/-3.4 mM (n=4). Besides, carticaine inhibits the phosphorylation of the enzyme by inorganic phosphate (Pi): Ki=20.0+/-3.4 (n=5) - 33.2+/-4.6 (n=4) mM, for [Pi] 1-4 mM. Carticaine increases the membrane permeability to Ca2+. Ca2+ efflux from preloaded vesicles is prevented by Ca2+ and Mg2+. Our results suggest that the diffusion of the local anesthetic into muscle fibers might trigger undesired effects such as sustained contraction of the masticatory muscles.

Calcium Release and Spread Within the Sarcomere of Vertebrate Skeletal Muscle Fibers.

Experiments were carried out to investigate excitation-contraction coupling in vertebrate skeletal muscle fibers. Single-dissected twitch fibers of frog muscle were injected with flourescent calcium (Ca) indicators, and fluorescence intensity (F) and changes in intensity (ΔF) were recorded during various stimulation protocols (16-22 °C). In fibers activated by an action potential, the low-affinity indicator furaptra was used to estimate the spatially-averaged change in myoplasmic free calcium concentration (ΔC[a]; peak value, 15-20 μM; time to peak, 4-5 ms). Δ[Ca], in combination with a kinetic model, was used to calculate the change in concentration of calcium bound to the principal myoplasmic Ca buffers (ATP, troponin, parvalbumin). The rate of Ca release from the sarcoplasmic reticulum (SR) was obtained from the time derivative of the estimated change in total myoplasmic calcium concentration, Δ[CaT] (= Δ[Ca] + Δ[CaATP] + Δ[CaTroponin] + Δ[CaParvalbumin]).

Identification of a contractile deficit in adult cardiac myocytes expressing hypertrophic cardiomyopathy-associated mutant troponin T proteins.

The direct effects of expressing hypertrophic cardiomyopathy-associated (HCM-associated) mutant troponin T (TnT) proteins on the force generation of single adult cardiac myocytes have not been established. Replication-defective recombinant adenovirus vectors were generated for gene transfer of HCM-associated I79N and R92Q mutant cardiac TnT cDNAs into fully differentiated adult cardiac myocytes in primary culture. We tested the hypothesis that the mutant TnT proteins would be expressed and incorporated into the cardiac sarcomere and would behave as dominant-negative proteins to directly alter calcium-activated force generation at the level of the single cardiac myocyte. Interestingly, under identical experimental conditions, the ectopic expression of the mutant TnTs was significantly less ( approximately 8% of total) than that obtained with expression of wild-type TnT ( approximately 35%) in the myocytes. Confocal imaging of immunolabeled TnT showed a regular periodic pattern of localization of ectopic mutant TnT that was not different than that in normal controls, suggesting that mutant TnT incorporation had no deleterious effects on sarcomeric architecture. Direct measurements of isometric force production in single cardiac myocytes demonstrated marked desensitization of submaximal calcium-activated tension, with unchanged maximum tension generation in mutant TnT-expressing myocytes compared with control myocytes. Collectively, these results demonstrate an impaired expression of the mutant protein and a disabling of cardiac contraction in the submaximal range of myoplasmic calcium concentrations. Our functional results suggest that development of new pharmacological, chemical, or genetic approaches to sensitize the thin-filament regulatory protein system could ameliorate force deficits associated with expression of I79N and R92Q in adult cardiac myocytes.

MYOPLASMIC CALCIUM CONTROL IN NORMAL HUMAN MUSCLE CELLS AND IN CELLS WITH AN ARG163CYS MUTATION OF THE RYR1 GENE

S318 INTRODUCTION: Molecular-genetic studies have identified the ryanodine receptor (RYR1) on chromosome 19 as the primary locus for malignant hyperthermia (MH) susceptibility. Mutation screening of the RYR1 gene in MH susceptible (MHS) individuals has identified 17 mutations [1] and hypersensitivity to halothane in primary human skeletal muscle cells has been shown for one known mutation of the RYR1 gene (Arg163Cys) [2]. The aim of the present study was to determine whether or not muscle cells of MH negative (MHN) subjects from unrelated families have reduced hypersensitivity compared with muscle cells from an individual with an Arg163Cys mutation. METHODS: MH screening was performed using the European protocol [3]. Primary skeletal muscle cell cultures were obtained from 9 MHN subjects and from one MHS subject with a known Arg163Cys mutation. Cells were cultured on glass coverslips and loaded with fura-2/AM. Effects of halothane on myoplasmic calcium concentration [Ca2+] were investigated at halothane concentrations of 3.9, 4.6, 5.7, 7.2 and 8.6 mM in 10 cells for each concentration and for all subjects. The increase of [Ca (2+)] is reported as increase of the fluorescence ratio of fura-2 measured at 340 nm and 380 nm and calculated on a pixel-by-pixel basis. RESULTS: Data are presented using a dose-response curve for cells with the Arg163Cys mutation and with connecting lines for each MHN subject. (Figure 1)Figure 1DISCUSSION: The increase of [Ca2+] to halothane challenges was decreased for all MHN cells compared with MHS cells carrying the Arg163Cys mutation. Our data support the view that a single RYR1 mutation may be causative for an MHS phenotype. Further investigations will show whether hypersensitivity to halothane may be also demonstrated in MHS cells with other RYR1 mutations.

Cardiac responses to calcium sensitizers and isoproterenol in intact guinea pig hearts. Effects on cyclic AMP levels, protein phosphorylation, myoplasmic calcium concentration, and left ventricular function.

Cardiac responses to calcium sensitizers and isoproterenol in intact guinea pig hearts. Effects on cyclic AMP levels, protein phosphorylation, myoplasmic calcium concentration, and left ventricular function.