Sarcoplasmic Calcium Release Research Articles

Enhanced capacity for CaMKII signaling mitigates calcium release related contractile fatigue with high intensity exercise

BackgroundWe tested whether enhancing the capacity for calcium/calmodulin-dependent protein kinase type II (CaMKII) signaling would delay fatigue of excitation-induced calcium release and improve contractile characteristics of skeletal muscle during fatiguing exercise. MethodsFast and slow type muscle, gastrocnemius medialis (GM) and soleus (SOL), of rats and mouse interosseus (IO) muscle fibers, were transfected with pcDNA3-based plasmids for rat α and β CaMKII or empty controls. Levels of CaMKII, its T287-phosphorylation (pT287-CaMKII), and phosphorylation of components of calcium release and re-uptake, ryanodine receptor 1 (pS2843-RyR1) and phospholamban (pT17-PLN), were quantified biochemically. Sarcoplasmic calcium in transfected muscle fibers was monitored microscopically during trains of electrical excitation based on Fluo-4 FF fluorescence (n = 5–7). Effects of low- (n = 6) and high- (n = 8) intensity exercise on pT287-CaMKII and contractile characteristics were studied in situ. ResultsCo-transfection with αCaMKII-pcDNA3/βCaMKII-pcDNA3 increased α and βCaMKII levels in SOL (+45.8 %, +250.5 %) and GM (+40.4 %, +89.9 %) muscle fibers compared to control transfection. High-intensity exercise increased pT287-βCaMKII and pS2843-RyR1 levels in SOL (+269 %, +151 %) and GM (+354 %, +119 %), but decreased pT287-αCaMKII and p17-PLN levels in GM compared to SOL (−76 % vs. +166 %; 0 % vs. +128 %). α/β CaMKII overexpression attenuated the decline of calcium release in muscle fibers with repeated excitation, and mitigated exercise-induced deterioration of rates in force production, and passive force, in a muscle-dependent manner, in correlation with pS2843-RyR1 and pT17-PLN levels (|r| > 0.7). ConclusionEnhanced capacity for α/β CaMKII signaling improves fatigue-resistance of active and passive contractile muscle properties in association with RyR1- and PLN-related improvements in sarcoplasmic calcium release.

Analysis of Drug Effects on iPSC Cardiomyocytes with Machine Learning

Patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offer an attractive experimental platform to investigate cardiac diseases and therapeutic outcome. In this study, iPSC-CMs were utilized to study their calcium transient signals and drug effects by means of machine learning, a central part of artificial intelligence. Drug effects were assessed in six iPSC-lines carrying different mutations causing catecholaminergic polymorphic ventricular tachycardia (CPVT), a highly malignant inherited arrhythmogenic disorder. The antiarrhythmic effect of dantrolene, an inhibitor of sarcoplasmic calcium release, was studied in iPSC-CMs after adrenaline, an adrenergic agonist, stimulation by machine learning analysis of calcium transient signals. First, beats of transient signals were identified with our peak recognition algorithm previously developed. Then 12 peak variables were computed for every identified peak of a signal and by means of this data signals were classified into different classes corresponding to those affected by adrenaline or, thereafter, affected by a drug, dantrolene. The best classification accuracy was approximately 79% indicating that machine learning methods can be utilized in analysis of iPSC-CM drug effects. In the future, data analysis of iPSC-CM drug effects together with machine learning methods can create a very valuable and efficient platform to individualize medication in addition to drug screening and cardiotoxicity studies.

Membrane and calcium clock mechanisms contribute variably as a function of temperature to setting cardiac pacemaker rate in zebrafish Danio rerio.

Here, we show that heart rate in zebrafish Danio rerio is dependent upon two pacemaking mechanisms and it possesses a limited ability to reset the cardiac pacemaker with temperature acclimation. Electrocardiogram recordings, taken from individual, anaesthetised zebrafish that had been acclimated to 18, 23 or 28°C were used to follow the response of maximum heart rate (fHmax ) to acute warming from 18°C until signs of cardiac failure appeared (up to c. 40°C). Because fHmax was similar across the acclimation groups at almost all equivalent test temperatures, warm acclimation was limited to one significant effect, the 23°C acclimated zebrafish had a significantly higher (21%) peak fHmax and reached a higher (3°C) test temperature than the 18°C acclimated zebrafish. Using zatebradine to block the membrane hyperpolarisation-activated cyclic nucleotide-gated channels (HCN) and examine the contribution of the membrane clock mechanisms to cardiac pacemaking, f Hmax was significantly reduced (by at least 40%) at all acute test temperatures and significantly more so at most test temperatures for zebrafish acclimated to 28°C vs. 23°C. Thus, HCN channels and the membrane clock were not only important, but could be modified by thermal acclimation. Using a combination of ryanodine (to block sarcoplasmic calcium release) and thapsigargin (to block sarcoplasmic calcium reuptake) to examine the contribution of sarcoplasmic reticular handling of calcium and the calcium clock, f Hmax was again consistently reduced independent of the test temperature and acclimation temperature, but to a significantly lesser degree than zatebradine for zebrafish acclimated to both 28 and 18°C. Thus, the calcium clock mechanism plays an additional role in setting pacemaker activity that was independent of temperature. In conclusion, the zebrafish cardiac pacemaker has a limited temperature acclimation ability compared with known effects for other fishes and involves two pacemaking mechanisms, one of which was independent of temperature.

Non-RyR Calcium Leak of the Sarcoplasmic Reticulum is Governed by TRPC1 in Cardiomyocytes

Sarcoplasmic calcium leak in cardiomyocytes has been explained by spontaneous sparks from ryanodine receptors (RyRs), non-spark-mediated RyR leak and non-RyR leak. Ion channels underlying non-RyR leak are currently ill defined. Here, we test the hypothesis that transient receptor potential cation (TRPC) 1 channels contribute to non-RyR leak. We investigated neonatal rat ventricular myocytes (NRVMs) infected with adenoviral eGFP, TRPC1-eGFP, shRNA-TRPC1-eGFP and scrambled RNA-eGFP constructs. NRVMs were studied 4-5 days after infection. NRVMs were fixed and labeled for TRPC1 and sarco/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2). Three-dimensional image stacks of NRVMs were acquired using a confocal microscope (Leica SP8 TCS). Protein colocalization was measured using the Pearson correlation coefficient (Rr). We also investigated SR calcium load in Rhod-3 loaded NRVMs. In short, NRVMs were paced and then superfused with 0 sodium/0 calcium solution for 2 min. Subsequently caffeine (20 mM) was applied to trigger sarcoplasmic calcium release. Fluorescence signals were acquired using a Leica SP8 TCS microscope and analysed after noise reduction and self-ratioing. We found a mid-level degree of colocalization of SERCA2 and TRPC1 for NRVMs infected with with eGFP (Rr=0.46±0.03), TRPC1-eGFP (0.41±0.03) and shRNA-TRPC1-eGFP (0.47±0.03). Colocalization of eGFP with SERCA2 was higher for TRPC1-eGFP (0.41±0.04) than for eGFP (0.26±0.02) and shRNA-TRPC1-eGFP infected NRVMs (0.22±0.04). We measured decreased and increased sarcoplasmic calcium content in NRVMs infected with TRPC1-eGFP (1.64±0.21) and shRNA-TRPC1-eGFP (4.30±0.49), respectively, versus eGFP (3.18±0.23). Differences between calcium content in eGFP and scrambled RNA-eGFP infected NRVMs were not significant. The investigations support our hypothesis that TRPC1 channels contribute to non-RyR sarcoplasmic calcium leak in cardiomyocytes. Structural data indicate that TRPC1 is located in the sarcoplasmic membrane. We suggest that expression and activity of TRPC1 channels modulate the concentration of sarcoplasmic and cytosolic calcium in cardiac myocytes.

SOCE Is Important for Maintaining Sarcoplasmic Calcium Content and Release in Skeletal Muscle Fibers

Store-operated Ca2+ entry (SOCE) is a Ca2+-entry process activated by the depletion of intracellular stores and has an important role in many cell types. In skeletal muscle, however, its role during physiological muscle activation has been controversial. To address this question, sarcoplasmic reticulum (SR) calcium release in a mouse strain with a naturally occurring mutation in the myostatin gene (Compact (Cmpt)) leading to a hypermuscular yet reduced muscle-force phenotype was compared to that in wild-type mice. To elicit Ca2+ release from the SR of flexor digitorum brevis (FDB) fibers, either a ryanodine receptor agonist (4-chloro-meta-cresol) or depolarizing pulses were used. In muscles from Cmpt mice, endogenous protein levels of STIM1 and Orai1 were reduced, and consequently, SOCE after 4-chloro-meta-cresol-induced store depletion was suppressed. Although the voltage dependence of SR calcium release was not statistically different between wild-type and Cmpt fibers, the amount of releasable calcium was significantly reduced in the latter, indicating a smaller SR content. To assess the immediate role of SOCE in replenishing the SR calcium store, the evolution of intracellular calcium concentration during a train of long-lasting depolarizations to a maximally activating voltage was monitored. Cmpt mice exhibited a faster decline in calcium release, suggesting a compromised ability to refill the SR. A simple model that incorporates a reduced SOCE as an important partner in regulating immediate calcium influx through the surface membrane readily accounts for the steady-state reduction in SR calcium content and its more pronounced decline after calcium release.

Dietary selenium augments sarcoplasmic calcium release and mechanical performance in mice.

BackgroundAs an essential trace element selenium plays a significant role in many physiological functions of the organs. It is found within muscles as selenocystein in selenoprotein N, which is involved in redox-modulated calcium homeostasis and in protection against oxidative stress.MethodsThe effects of two different selenium compounds (selenate and NanoSe in 0.5 and 5 ppm concentration for two weeks) on muscle properties of mice were examined by measuring in vivo muscle performance, in vitro force in soleus (SOL) and extensor digitorum longus (EDL) muscles and changes in intracellular Ca2+ concentration in single fibers from flexor digitorum brevis (FDB) muscle.. Western-blot analysis on muscle lysates of EDL and SOL were used to measure the selenoprotein N expression. Control mice received 0.3 ppm Se.ResultsWhile the grip force did not change, 5 ppm selenium diets significantly increased the speed of voluntary running and the daily distance covered. Both forms of selenium increased significantly the amplitude of single twitches in EDL and SOL muscle in a concentration dependent manner. Selenate increased fatigue resistance in SOL. The amplitude of the calcium transients evoked by KCl depolarization increased significantly from the control of 343 ± 44 nM to 671 ± 51 nM in the presence of 0.5 ppm selenate in FDB fibers. In parallel, the rate of calcium release during short depolarizations increased significantly from 28.4 ± 2.2 to 45.5 ± 3.8 and 52.1 ± 1.9 μM/ms in the presence of 0.5 ppm NanoSe and selenate, respectively. In 0.5 ppm concentration both selenium compounds increased significantly the selenoprotein N expression only in EDL muscle.ConclusionsSelenium supplementation augments calcium release from the sarcoplasmic reticulum thus improves skeletal muscle performance. These effects are accompanied by the increased selenoprotein N expression in the muscles which could result in increased oxidative stress tolerance in case of long lasting contraction.Electronic supplementary materialThe online version of this article (doi:10.1186/s12986-016-0134-6) contains supplementary material, which is available to authorized users.

Modification of Bupivacaine-Induced Myotoxicity with Dantrolene and Caffeine In Vitro.

Local anesthetics, especially bupivacaine, have myotoxic effects in clinically used concentrations and context. Detailed mechanisms of these effects are unknown, but an increase in intracellular calcium levels is suspected to be the most important trigger. Dantrolene and caffeine modify cellular calcium release from the sarcoplasmic reticulum. The aim of our study was to investigate the effect of dantrolene and caffeine on bupivacaine-induced myotoxicity in vitro. A cell culture model of primary muscle cells of BALB/c AnNCrl mice was established. Cells were incubated simultaneously with increasing concentrations of bupivacaine, dantrolene, and caffeine. The fraction of dead cells was calculated after staining with propidium iodide and analysis by flow cytometry. The half-maximal inhibitory concentration of bupivacaine was calculated for each concentration. Group differences were determined by using 1-way analysis of variances with subsequent post hoc 1-way Dunnett t test. Both dantrolene and caffeine alone had no effect on muscle cell survival. Increasing concentrations of bupivacaine caused increasing cell death. Dantrolene dose-dependently reduced the fraction of necrotic cells, whereas caffeine dose-dependently increased the fraction of dead cells. Dantrolene attenuated, and caffeine enhanced, bupivacaine-induced myotoxicity, presumably by modifying sarcoplasmic calcium release. This indicates that intracellular calcium release is an important factor for local anesthetic-induced cell death.

Management of malignant hyperthermia: diagnosis and treatment.

Malignant hyperthermia is a potentially lethal inherited disorder characterized by disturbance of calcium homeostasis in skeletal muscle. Volatile anesthetics and/or the depolarizing muscle relaxant succinylcholine may induce this hypermetabolic muscular syndrome due to uncontrolled sarcoplasmic calcium release via functionally altered calcium release receptors, resulting in hypoxemia, hypercapnia, tachycardia, muscular rigidity, acidosis, hyperkalemia, and hyperthermia in susceptible individuals. Since the clinical presentation of malignant hyperthermia is highly variable, survival of affected patients depends largely on early recognition of the symptoms characteristic of malignant hyperthermia, and immediate action on the part of the attending anesthesiologist. Clinical symptoms of malignant hyperthermia, diagnostic criteria, and current therapeutic guidelines, as well as adequate management of anesthesia in patients susceptible to malignant hyperthermia, are discussed in this review.

Overexpression of atrial adenosine2A receptors induced by high-rate pacing stimulation: a new step in the molecular remodeling of the atria

Recently, an association between atrial fibrillation (AF) and a local increase in sarcoplasmic calcium release was suggested as a mechanism able to generate afterdepolarizations and promote arrhythmias recurrence. The adenosine A2A receptors (A2A) are expressed in the atria, modulate the frequency of spontaneous calcium release, and may be involved in the remodeling process during AF. Aim: To investigate the role of atrial adenosine receptors by analyzing A1 and A2A receptor protein levels through changes in their transcriptional control during atrial high-rate. Methods: The high-right atrium of anesthetized, artificially ventilated rats (n=10; age 12 weeks) was paced epicardially for 2 hours (50Hz). Animals were sacrificed and separate samples of the atria collected for A1 and A2A mRNA Real Time PCR quantification. The housekeeping gene 18SrRNA was used as internal control for the quantification of receptors mRNA. A sham group was used as control (matching number, age and sex). CT values were calculated for each sample, and converted into relative quantities with the ΔCTmethod (Eq.1: ΔCT=CTAdora-CT18SrRNA; Eq.2: ΔΔCT=ΔCTcontrols- ΔCTStimulation). The effects of high-rate pacing were evaluated by one way analysis of variance (significance if P<0.05). Results: After 2 hours of high-rate pacing, real time PCR showed a significant increase of A2A mRNA expression levels (ΔCTA2A 16,8±2.72 vs. 11,2±2.64; p<0.001), and a steady maintenance of the A1 mRNA levels. No differences between right and left atrium were observed. ![Figure][1] Figure 1. Adenosine receptors remodeling Conclusions: Sustained atrial high-rate stimulation induces an early overexpression of A2A receptors, suggesting an impairment of the cross-talk between atrial A1 and A2A receptors. This can be associated with a role for A2A receptors in atrial Ca2+ kinetics and atrial remodeling. [1]: pending:yes

Trypanosoma brucei cathepsin-L increases arrhythmogenic sarcoplasmic reticulum-mediated calcium release in rat cardiomyocytes

AimsAfrican trypanosomiasis, caused by Trypanosoma brucei species, leads to both neurological and cardiac dysfunction and can be fatal if untreated. While the neurological-related pathogenesis is well studied, the cardiac pathogenesis remains unknown. The current study exposed isolated ventricular cardiomyocytes and adult rat hearts to T. brucei to test whether trypanosomes can alter cardiac function independent of a systemic inflammatory/immune response.Methods and resultsUsing confocal imaging, T. brucei and T. brucei culture media (supernatant) caused an increased frequency of arrhythmogenic spontaneous diastolic sarcoplasmic reticulum (SR)-mediated Ca2+ release (Ca2+ waves) in isolated adult rat ventricular cardiomyocytes. Studies utilising inhibitors, recombinant protein and RNAi all demonstrated that this altered SR function was due to T. brucei cathepsin-L (TbCatL). Separate experiments revealed that TbCatL induced a 10–15% increase of SERCA activity but reduced SR Ca2+ content, suggesting a concomitant increased SR-mediated Ca2+ leak. This conclusion was supported by data demonstrating that TbCatL increased Ca2+ wave frequency. These effects were abolished by autocamtide-2-related inhibitory peptide, highlighting a role for CaMKII in the TbCatL action on SR function. Isolated Langendorff perfused whole heart experiments confirmed that supernatant caused an increased number of arrhythmic events.ConclusionThese data demonstrate for the first time that African trypanosomes alter cardiac function independent of a systemic immune response, via a mechanism involving extracellular cathepsin-L-mediated changes in SR function.

Rocuronium and malignant hyperthermia

Beggs et al.1 presented two interesting case reports postulating an association between rocuronium and malignant hyperthermia (MH). Although the observed fever in these patients declined after discontinuation of rocuronium, several points need further discussion. First, the definition of MH includes hypercapnia, lactic acidosis, and skeletal muscle hypermetabolism due to excessive sarcoplasmic calcium release via mutated ryanodine receptors after exposure to triggering agents.2 As both patients had complex medical conditions (e.g., acute respiratory distress syndrome, history of cardiopulmonary resuscitation, sepsis), disorders such as metabolic acidosis might have occurred for various reasons. Although MH certainly could have been one reason, this seems rather unlikely since the acidosis was not even temporally related to the use of rocuronium. Second, the body temperature in the reported cases remained elevated for several days, which is not typical of MH crisis. Even though there have been several reports of MH with hyperthermia as the only symptom,3 other findings, such as the modest creatine kinase elevation (351 units/L in patient 1), are inconsistent with a diagnosis of MH. Furthermore, unexplained fever among patients in the intensive care unit is very common and associated with a large variety of differential diagnoses.4

In Vitro Contracture Test Results and Anaesthetic Management of a Patient with Emery-Dreifuss Muscular Dystrophy for Cardiac Transplantation

Emery-Dreifuss muscular dystrophy (EDMD) is a hereditary neuromuscular disorder characterized by slowly progressive muscle weakness, early contractures, and dilated cardiomyopathy. We reported an uneventful general anaesthesia using total intravenous anaesthesia (TIVA) for cardiac transplantation in a 19-year-old woman suffering from EDMD. In vitro contracture test results of two pectoralis major muscle bundles of the patient suggest that exposition to triggering agents does not induce a pathological sarcoplasmic calcium release in the lamin A/C phenotype. However, due to the lack of evidence in the literature, we would recommend TIVA for patients with EDMD if general anaesthesia is required.

Maligne Hyperthermie – Diagnostik, Therapie und Narkoseführung

In malignant hyperthermia (MH) susceptible individuals volatile anaesthetics and the depolarizing muscle relaxant succinylcholine may induce a potentially lethal hypermetabolic syndrome of skeletal muscle due to an uncontrolled sarcoplasmic calcium release. Immediate discontinuation of triggering agents, oxygenation, correction of acidosis and electrolyte abnormalities and dantrolene application are essential for MH treatment. This article reviews the clinical symptoms of MH, the diagnostic criteria and the actual guidelines for treatment and management of anaesthesia in susceptible individuals.

Clues to the Formation of Cores in a Mouse Model of Malignant Hyperthermia

Malignant hyperthermia (MH) and central core disease (CCD) are closely related diseases of skeletal muscle linked to mutations in the ryanodine receptor (RyR1) gene. Heterozygous mice harbouring a human MH mutation (Y522S), which is linked to MH susceptibility (MHS) with cores, were shown to display MH susceptibility that is also associated with heat-induced sudden death and mitochondrial damage (Durham et al., 2008; Cell: 133, 53). Here we show that RyR1Y522S/wt fibers develop amorphous cores which mimic, at least in later stages, the structural alterations observed in muscle biopsies from CCD patients. By examining mice at various ages (2 m - 1 y), we identified early steps in the formation of cores, a feat that has not been possible in human CCD muscle. The earliest and most obvious event in “core” formation is the swelling and partial disruption of mitochondria within discrete regions of the cell. This defect quickly leads to disarray of closely associated sarcoplasmic reticulum (SR) and calcium release units (CRUs), ultimately leading to the eventual complete disorganization of both organelles. The core regions also exhibit clearly delimited myofibril contractures, probably due increased Ca2+ leak from the SR diffusing from adjacent regions that cannot be sequestered. In later stages, amorphous cores, lacking all structural components become more frequent. We suggest that the initial triggering event in core formation is a local imbalance in Ca2+ release due to increased RyR1 leak that initially alters mitochondrial activity and then eventually disrupts both mitochondria and SR structure/function. Loss of mitochondrial ATP production and SR Ca2+ sequestration in these regions leads to local contractures and sarcomeric disruption within the core regions.

Myosin heavy chain isoform content and energy metabolism can be uncoupled in pig skeletal muscle

Genetic selection for improved growth and overall meatiness has resulted in the occurrence of 2 major mutations in pigs, the Rendement Napole (RN) and Halothane (Hal) gene mutations. At the tissue level, these mutations influence energy metabolism in skeletal muscle and muscle fiber type composition, yet also influence total body composition. The RN mutation affects the adenosine monophosphate-activated protein kinase gamma subunit and results in increased glycogen deposition in the muscle, whereas the Hal mutation alters sarcoplasmic calcium release mechanisms and results in altered energy metabolism. From a meat quality standpoint, these mutations independently influence the extent and rate of muscle energy metabolism postmortem, respectively. Even though these mutations alter overall muscle energy metabolism and histochemically derived muscle fiber type independently, their effects have not been yet fully elucidated in respect to myosin heavy chain (MyHC) isoform content and those enzymes responsible for defining energetics of the tissue. Therefore, the objective of this study was to determine the collective effects of the RN and Hal genes on genes and gene products associated with different muscle fiber types in pig skeletal muscle. To overcome potential pitfalls associated with traditional muscle fiber typing, real-time PCR, gel electrophoresis, and Western blotting were used to evaluate MyHC composition and several energy-related gene expressions in muscles from wild-type, RN, Hal, and Hal-RN mutant pigs. The MyHC mRNA levels displayed sequential transitions from IIb to IIx and IIa in pigs bearing the RN mutation. In addition, our results showed MyHC protein isoform abundance is correlated with mRNA level supporting the hypothesis that MyHC genes are transcriptionally controlled. However, transcript abundance of genes involved in energy metabolism, including lactate dehydrogenase, citrate synthase, glycogen synthase, and peroxisome proliferator-activated receptor alpha, was not different between genotypes. These data show that the RN and Hal gene mutations alter muscle fiber type composition and suggest that muscle fiber energy metabolism and speed of contraction, the 2 determinants of muscle fiber type, can be uncoupled.

Paraneoplastic myasthenia gravis: immunological and clinical aspects

Paraneoplastic myasthenia gravis (MG) is accompanied by a neoplasm, usually thymoma. In patients with thymoma and a specific genetic make-up, the paraneoplastic immune response develops further in thymic remnant or peripheral lymphatic tissue. Paraneoplastic MG and late-onset MG (age >or= 50 years) share a similar immunological profile with high titin and ryanodine receptor (RyR) antibody prevalence. This profile is the most important predictor of clinical outcome in paraneoplastic MG. The presence of a thymoma per se does not cause more severe MG. MG severity is linked to the patient's immunological profile. Paraneoplastic MG causes a distinctive non-limb symptom profile at MG onset, characterized by bulbar, ocular, neck, and respiratory symptoms. When the diagnosis of paraneoplastic MG is established, the neoplasm should be removed surgically. Pre-thymectomy plasmapheresis or iv-IgG should be considered in these patients to minimize post-thymectomy MG exacerbation risk. Paraneoplastic MG usually continues after thymectomy. The pharmacological treatment of paraneoplastic MG does not differ from non-paraneoplastic MG, except for tacrolimus that should be considered in difficult cases. Tacrolimus is an immunosuppressant acting specifically in RyR antibody positive patients through enhancing RyR-related sarcoplasmic calcium release that in theory might be blocked by RyR antibodies, causing symptomatic relief in paraneoplastic MG.

Not Every Hypermetabolic State Is Due to Malignant Hyperthermia!

To the Editor: We have concerns about the diagnosis of malignant hyperthermia (MH) in a recent case report by Lichtman and Oribabor (1). As the authors stated “several items in this patient’s postoperative course were atypical ….” One hour postoperatively the patient developed a hypermetabolic state with combined respiratory and metabolic acidosis. This state was successfully treated with dantrolene. The fact that dantrolene was effective is not surprising. Muscle rigidity from any cause places considerable metabolic demand on the body, which is effectively attenuated by blocking sarcoplasmic calcium release by dantrolene. Indeed, dantrolene is effective in other hypermetabolic diseases, such as malignant neuroleptic syndrome (2) and is an effective therapeutic strategy to reduce muscle protein breakdown and muscle lactate formation (3). Despite the potentially positive family history and muscle rigidity, there were no other clinical signs of MH. There are many reasons for postoperative hypermetabolic states, e.g., sepsis, hypoperfusion, and propofol infusion syndrome (4–6). It would have been useful had the authors discussed alternatives and not focused on only one possible diagnosis. MH is defined as a pharmacogenetic disease, where triggering agents (volatile anesthetic agents and depolarizing neuromuscular blocking agents) initiate a hypermetabolic reaction in genetically susceptible patients. This definition separates MH from other hypermetabolic states. We do not question the choice of dantrolene for this patient, although we are not aware of the evidence to initiate an oral dantrolene therapy. We similarly are unaware of evidence to support the conclusion to consider off-pump CABG rather than traditional hypothermic CPB in MH susceptible individuals. Lastly, although we respect the patient’s decision to refuse testing for MH, we are disappointed that further testing could not be done. Molecular genetic investigations are now available in North America (7) and alternative minimal invasive methods have been recently presented (8). It is particularly odd that patient without symptomatic cardiac disease would agree to coronary bypass grafting in order to improve his preoperative condition for the planned shoulder surgery, but refuse testing for MH, and why the currently proposed diagnostic approach for MH testing has not been applied to this patient and his family (7). Thierry Girard, MD Albert Urwyler, MD Department of Anesthesia University Hospital Basel CH 4031 Basel Switzerland [email protected]

RyR isoforms and fibre type-specific expression of proteins controlling intracellular calcium concentration in skeletal muscles

Muscle fibres which shorten with high maximum shortening velocity also exhibit fast kinetics of contraction, i.e. short values of time to peak tension and time to half relaxation. This short review aims to discuss the molecular basis of such correlation, to reach, based on the available literature, an answer to the question whether there is a correlation in expression of proteins determining shortening velocity, myosin isoforms in the first place, and proteins controlling cytosolic calcium concentration and its variations at rest or during contraction. Although the isoforms of RyR, the sarcoplasmic calcium release channels, do not show a tightly coordinated expression with myosin isoforms, other proteins involved in controlling intracellular calcium do. This is likely sufficient to guarantee the correlation between maximum shortening velocity and speed of isometric contraction.

Nitric oxide modifies the sarcoplasmic reticular calcium release channel in endotoxemia by both guanosine-3′,5′ (cyclic) phosphate-dependent and independent pathways*

a) To determine whether decreased sarcoplasmic calcium release channel (CRC) activity is a mechanism by which myocardial contractility is reduced in endotoxemia; b) to determine whether nitric oxide modulates CRC activity in endotoxemia; and c) to examine two nitric oxide signaling pathways in relation to CRC function in endotoxemia. Randomized, prospective using a rat model of endotoxemia. : Research laboratory. Sprague-Dawley rats. Endotoxemia was induced by lipopolysaccharide administration. The effects of nitric oxide were studied using the highly selective inducible nitric oxide synthase inhibitor N-(3-(aminomethyl)benzyl)acetamidine dihydrochloride (1400W) and the specific guanylyl cyclase inhibitor 1-H (1, 2, 4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). We assessed myocardial contractility, myocardial nitric oxide content, and guanosine-3',5' (cyclic) phosphate (cGMP) content. We determined CRC activity by calcium release and ryanodine binding assays. We followed these variables at four time points through the course of endotoxemia. We found that myocardial contractility and CRC activity were decreased in late but not in early endotoxemia. Furthermore, inducible nitric oxide synthase inhibition with 1400W restored contractility and CRC activity in late endotoxemia but paradoxically worsened these variables in early endotoxemia. Through the use of the guanylyl cyclase inhibitor ODQ, we demonstrate that nitric oxide acts through cGMP-mediated mechanisms in early and late endotoxemia. We investigated cGMP-independent pathways by assessing the oxidative status of the CRC. We found that in late endotoxemia, nitric oxide decreased the number of free thiols, demonstrating that nitric oxide also acts through cGMP-independent pathways. Nitric oxide has a dual effect on the CRC in endotoxemia. At low concentrations, as measured in early endotoxemia, nitric oxide stabilizes the CRC through cGMP-mediated mechanisms. In late endotoxemia, high nitric oxide concentrations decrease channel activity through both cGMP-dependent and cGMP-independent mechanisms.

Nitric oxide affects sarcoplasmic calcium release in skeletal myotubes.

In the present study, we used real-time confocal microscopy to examine the effects of two nitric oxide (NO) donors on acetylcholine (ACh; 10 microM)- and caffeine (10 mM)-induced intracellular calcium concentration ([Ca2+]i) responses in C2C12 mouse skeletal myotubes. We hypothesized that NO reduces [Ca2+]i in activated skeletal myotubes through oxidation of thiols associated with the sarcoplasmic reticulum Ca2+-release channel. Exposure to diethylamine NONOate (DEA-NO) reversibly increased resting [Ca2+]i level and resulted in a dose-dependent reduction in the amplitude of ACh-induced [Ca2+]i responses (25 +/- 7% reduction with 10 microM DEA-NO and 78 +/- 14% reduction with 100 microM DEA-NO). These effects of DEA-NO were partly reversible after subsequent exposure to dithiothreitol (10 mM). Preexposure to DEA-NO (1, 10, and 50 microM) also reduced the amplitude of the caffeine-induced [Ca2+]i response. Similar data were obtained by using the chemically distinct NO donor S-nitroso-N-acetyl-penicillamine (100 microM). These results indicate that NO reduces sarcoplasmic reticulum Ca2+ release in skeletal myotubes, probably by a modification of hyperreactive thiols present on the ryanodine receptor channel.