Progressive impairment of CaV1.1 function in the skeletal muscle of mice expressing a mutant type 1 Cu/Zn superoxide dismutase (G93A) linked to amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder that is typically fatal within 3-5 years of diagnosis. While motoneuron death is the defining characteristic of ALS, the events that underlie its pathology are not restricted to the nervous system. In this regard, ALS muscle atrophies and weakens significantly before presentation of neurological symptoms. Since the skeletal muscle L-type Ca(2+) channel (CaV1.1) is a key regulator of both mass and force, we investigated whether CaV1.1 function is impaired in the muscle of two distinct mouse models carrying an ALS-linked mutation. METHODS: We recorded L-type currents, charge movements, and myoplasmic Ca(2+) transients from dissociated flexor digitorum brevis (FDB) fibers to assess CaV1.1 function in two mouse models expressing a type 1 Cu/Zn superoxide dismutase mutant (SOD1(G93A)). RESULTS: In FDB fibers obtained from "symptomatic" global SOD1(G93A) mice, we observed a substantial reduction of SR Ca(2+) release in response to depolarization relative to fibers harvested from age-matched control mice. L-type current and charge movement were both reduced by ~40 % in symptomatic SOD1(G93A) fibers when compared to control fibers. Ca(2+) transients were not significantly reduced in similar experiments performed with FDB fibers obtained from "early-symptomatic" SOD1(G93A) mice, but L-type current and charge movement were decreased (~30 and ~20 %, respectively). Reductions in SR Ca(2+) release (~35 %), L-type current (~20 %), and charge movement (~15 %) were also observed in fibers obtained from another model where SOD1(G93A) expression was restricted to skeletal muscle. CONCLUSIONS: We report reductions in EC coupling, L-type current density, and charge movement in FDB fibers obtained from symptomatic global SOD1(G93A) mice. Experiments performed with FDB fibers obtained from early-symptomatic SOD1(G93A) and skeletal muscle autonomous MLC/SOD1(G93A) mice support the idea that events occurring locally in the skeletal muscle contribute to the impairment of CaV1.1 function in ALS muscle independently of innervation status.

Rem uncouples excitation-contraction coupling in adult skeletal muscle fibers.

In skeletal muscle, excitation-contraction (EC) coupling requires depolarization-induced conformational rearrangements in L-type Ca(2+) channel (Ca(V)1.1) to be communicated to the type 1 ryanodine-sensitive Ca(2+) release channel (RYR1) of the sarcoplasmic reticulum (SR) via transient protein-protein interactions. Although the molecular mechanism that underlies conformational coupling between Ca(V)1.1 and RYR1 has been investigated intensely for more than 25 years, the question of whether such signaling occurs via a direct interaction between the principal, voltage-sensing α(1S) subunit of Ca(V)1.1 and RYR1 or through an intermediary protein persists. A substantial body of evidence supports the idea that the auxiliary ß(1a) subunit of Ca(V)1.1 is a conduit for this intermolecular communication. However, a direct role for ß(1a) has been difficult to test because ß(1a) serves two other functions that are prerequisite for conformational coupling between Ca(V)1.1 and RYR1. Specifically, ß(1a) promotes efficient membrane expression of Ca(V)1.1 and facilitates the tetradic ultrastructural arrangement of Ca(V)1.1 channels within plasma membrane-SR junctions. In this paper, we demonstrate that overexpression of the RGK protein Rem, an established ß subunit-interacting protein, in adult mouse flexor digitorum brevis fibers markedly reduces voltage-induced myoplasmic Ca(2+) transients without greatly affecting Ca(V)1.1 targeting, intramembrane gating charge movement, or releasable SR Ca(2+) store content. In contrast, a ß(1a)-binding-deficient Rem triple mutant (R200A/L227A/H229A) has little effect on myoplasmic Ca(2+) release in response to membrane depolarization. Thus, Rem effectively uncouples the voltage sensors of Ca(V)1.1 from RYR1-mediated SR Ca(2+) release via its ability to interact with ß(1a). Our findings reveal Rem-expressing adult muscle as an experimental system that may prove useful in the definition of the precise role of the ß(1a) subunit in skeletal-type EC coupling.

Apparent lack of physical or functional interaction between CaV1.1 and its distal C terminus.

CaV1.1 acts as both the voltage sensor that triggers excitation-contraction coupling in skeletal muscle and as an L-type Ca(2+) channel. It has been proposed that, after its posttranslational cleavage, the distal C terminus of CaV1.1 remains noncovalently associated with proximal CaV1.1, and that tethering of protein kinase A to the distal C terminus is required for depolarization-induced potentiation of L-type Ca(2+) current in skeletal muscle. Here, we report that association of the distal C terminus with proximal CaV1.1 cannot be detected by either immunoprecipitation of mouse skeletal muscle or by colocalized fluorescence after expression in adult skeletal muscle fibers of a CaV1.1 construct labeled with yellow fluorescent protein (YFP) and cyan fluorescent protein on the N and C termini, respectively. We found that L-type Ca(2+) channel activity was similar after expression of constructs that either did (YFP-CaV1.11860) or did not (YFP-CaV1.11666) contain coding sequence for the distal C-terminal domain in dysgenic myotubes null for endogenous CaV1.1. Furthermore, in response to strong (up to 90 mV) or long-lasting prepulses (up to 200 ms), tail current amplitudes and decay times were equally increased in dysgenic myotubes expressing either YFP-CaV1.11860 or YFP-CaV1.11666, suggesting that the distal C-terminal domain was not required for depolarization-induced potentiation. Thus, our experiments do not support the existence of either biochemical or functional interactions between proximal CaV1.1 and the distal C terminus.

Functional assessment of three Rem residues identified as critical for interactions with Ca(2+) channel ß subunits.

Members of the Rem, Rem2, Rad, Gem/Kir (RGK) family of small GTP-binding proteins inhibit high-voltage-activated (HVA) Ca(2+) channels through interactions with both the principal α1 and the auxiliary ß subunits of the channel complex. Three highly conserved residues of Rem (R200, L227, and H229) have been shown in vitro to be critical for interactions with ß subunits. However, the functional significance of these residues is not known. To investigate the contributions of R200, L227, and H229 to ß subunit-mediated RGK protein-dependent inhibition of HVA channels, we introduced alanine substitutions into all three positions of Venus fluorescent protein-tagged Rem (V-Rem AAA) and made three other V-Rem constructs with an alanine introduced at only one position (V-Rem R200A, V-Rem L227A, and V-Rem H229A). Confocal imaging and immunoblotting demonstrated that each Venus-Rem mutant construct had comparable expression levels to Venus-wild-type Rem when heterologously expressed in tsA201 cells. In electrophysiological experiments, V-Rem AAA failed to inhibit N-type Ca(2+) currents in tsA201 cells coexpressing CaV2.2 α1B, ß3, and α2δ-1 channel subunits. The V-Rem L227A single mutant also failed to reduce N-type currents conducted by coexpressed CaV2.2 channels, a finding consistent with the previous observation that a leucine at position 227 is critical for Rem-ß interactions. Rem-dependent inhibition of CaV2.2 channels was impaired to a much lesser extent by the R200A substitution. In contrast to the earlier work demonstrating that Rem H229A was unable to interact with ß3 subunits in vitro, V-Rem H229A produced nearly complete inhibition of CaV2.2-mediated currents.

[Impact of a program designed to improve continuous renal replacement therapy stability]. / Impact d'un programme d'amélioration de la stabilité de l'épuration extrarénale continue.

OBJECTIVES: During continuous renal replacement therapy (CRRT), circuit clotting increases nursing workload, cost of the therapy and blood loss. The aim of this study was to assess the impact of a program designed to improve CRRT stability on unexpected circuit clotting. STUDY DESIGN: Retrospective and observational study. PATIENTS AND METHODS: In January 2011, several changes have been adopted regarding CRRT management. Regional citrate anticoagulation, continuous hemodialysis using super high-flux membranes and a specific training for intensive care unit nurses were implemented. CRRT sessions before (year 2009 and 2010, "Before group") and after (year 2011 and 2012, "After group") were analyzed. The primary endpoint was the incidence of unexpected CRRT session end. RESULTS: During the study period, 401 sessions performed in 152 patients were analyzed. Sixty-three unexpected session's end (40%) occurred before and 43 (17%) after the implementation of the program (P<0.0001). Median filter life time was 33 (13-48) hours before and 55 (27-67) hours after (P<0.0001). CONCLUSION: Our program designed to improve CRRT stability reduced filter losses by reducing unexpected circuit clotting.

[The acute pulmonary renal syndrome: An unusual presentation of granulomatosis with polyangiitis]. / Le syndrome pneumo-rénal aigu : un mode de révélation rare de la granulomatose avec polyangéite.

INTRODUCTION: We report a case of acute pulmonary renal syndrome mimicking septic shock, which led to the diagnosis of granulomatosis with polyangiitis. CASE REPORT: A 70-year-old man was hospitalized because of acute kidney injury and acute respiratory distress syndrome with diffuse alveolar hemorrhage associated with a serum procalcitonin level of 18 µg/L. Initially, septic shock was suspected and antibiotic therapy was started. The absence of microbiological isolates and the patient's rapid clinical deterioration prompted laboratory testing for autoimmune disease, which confirmed the diagnosis of granulomatosis with polyangiitis. Immunosuppressive therapy was promptly initiated with corticosteroids, cyclophosphamide and several plasma exchanges, which resulted in a rapid clinical improvement and ICU discharge. CONCLUSIONS: Granulomatosis with polyangiitis is a systemic necrotizing vasculitis with antineutrophil cytoplasmic antibodies, which can present with acute pulmonary renal syndrome, combining acute respiratory distress syndrome and acute kidney injury. This misleading presentation must prompt an autoimmune disease testing in order to yield an early diagnosis of a vasculitis, allowing for timely initiation of immunosuppressive treatment. Serum procalcitonin levels can be markedly elevated and this must not override the possibility of a vasculitis where the patient shows a compatible symptomatology.

RGK protein-mediated impairment of slow depolarization- dependent Ca2+ entry into developing myotubes.

Three physiological functions have been described for the skeletal muscle 1,4-dihydropyridine receptor (Ca(V)1.1):(1) voltage-sensor for excitation-contraction (EC) coupling, (2) L-type Ca(2+) channel, and (3) voltage-sensor for slow depolarization-dependent Ca(2+) entry. Members of the RGK (Rad, Rem, Rem2, Gem/Kir) family of monomeric GTP-binding proteins are potent inhibitors of the former two functions of Ca(V)1.1. However, it is not known whether the latter function that has been attributed to Ca(V)1.1 is subject to modulation by RGK proteins. Thus, the purpose of this study was to determine whether Rad, Gem and/or Rem inhibit the slowly developing, persistent Ca(2+) entry that is dependent on the voltage-sensing capability of Ca(V)1.1. As a means to investigate this question, Venus fluorescent protein-fused RGK proteins(V-Rad, V-Rem and V-Gem) were overexpressed in "normal" mouse myotubes. We observed that such overexpression of V-Rad, V-Rem or V-Gem in myotubes caused marked changes in morphology of the cells. As shown previously for YFPRem,both L-type current and EC coupling were also impaired greatly in myotubes expressing either V-Rad or V-Gem. There ductions in L-type current and EC coupling were paralleled by reductions in depolarization-induced Ca(2+) entry. Our observations provide the first evidence of modulation of this enigmatic Ca(2+) entry pathway peculiar to skeletal muscle.

Immediate allergic cross-reactivity to levobupivacaine and ropivacaine.

Allergic reactions to amide local anaesthetic agents are rare. We report the case of a 74-year-old man who suffered anaphylaxis, presenting with cardiovascular collapse, immediately after receiving regional anaesthesia on two separate occasions, the first involving the use of levobupivacaine and the second using ropivacaine. Skin testing revealed positive reactions to both levobupivacaine and ropivacaine, and negative reactions to articaine and lidocaine. Severe allergic reactions can be caused by the amide local anaesthetic drugs, levobupivacaine and ropivacaine.

Binding of FoxM1 to G2/M gene promoters is dependent upon B-Myb.

The promoters of genes which regulate entry into and progress through mitosis are typically induced maximally in G2 by transcription factors that include B-Myb and FoxM1. As FoxM1 gene transcription is a target of B-Myb, we investigated in this study how these transcription factors functionally interact to regulate these G2/M genes. Using a 3T3 cell line containing floxed B-myb alleles (B-myb(F/F)) that could be conditionally deleted by Cre recombinase, we confirmed that B-myb knockout caused both decreased mRNA expression of several G2/M genes, including FoxM1, and delayed entry into mitosis. Although FoxM1 protein expression was actually unaffected by B-myb knockout when quiescent B-myb(F/F) 3T3 cells re-entered the cell cycle upon serum-stimulation, chromatin immunoprecipitation revealed that FoxM1 binding to G2/M promoters was substantially reduced. FoxM1 transcriptional activity requires sequential phosphorylation by Cyclin-dependent kinases and Plk1, which are B-Myb target genes, and we found that phosphorylation at Plk1-specific sites was somewhat reduced upon B-myb knockout. Neither this effect nor nuclear accumulation of FoxM1, which was unaffected by B-myb knockout, was sufficient to account for the dependence on B-Myb for FoxM1 promoter binding, however. More significantly, assays using paired Birc5 (survivin) promoter-luciferase reporters with either wild-type or mutated Myb binding sites showed that FoxM1 was unable to bind and activate the promoter in the absence of B-Myb binding. Our data suggest that B-Myb is required as a pioneer factor to enable FoxM1 binding to G2/M gene promoters and explains how these transcription factors may collaborate to induce mitosis.

Live imaging of bulk endocytosis in frog motor nerve terminals using FM dyes.

We observed endocytosis in real time in stimulated frog motor nerve terminals by imaging the growth of large membrane infoldings labeled with a low concentration of FM dye. The spatial and temporal information made available by these experiments allowed us to image several new aspects of this synaptic vesicle recycling pathway. Membrane infoldings appeared near synaptic vesicle clusters and grew rapidly during long-duration, high-frequency stimulation. In some cases, we observed large, elongated infoldings growing laterally into the terminal. We used these observations to calculate infolding growth rates. A decrease in stimulation frequency caused a decrease in growth rates, but the overall length of these structures was unaffected by frequency changes. Attempts to wash the dye from these infoldings after stimulation were unsuccessful, demonstrating that the fluorescent structures had been endocytosed. We also used this technique to trigger and image infoldings during repeated, short trains. We found that membrane uptake occurred repeatedly at individual endocytosis sites, but only during a portion of the total number of trains delivered to the terminal. Finally, we showed that phosphatidylinositol 3-kinase, but not actin, was involved in this endocytosis pathway. The ability to monitor many individual bulk endocytosis sites in real time should allow for new types of endocytosis measurements and could reveal novel and unexpected mechanisms for coordinating membrane recovery during synaptic activity.

[Posterior reversible encephalopathy related to tacrolimus in a liver transplanted HIV patient]. / Encéphalopathie postérieure réversible liée au tacrolimus chez un patient transplanté hépatique infecté par le VIH.

Tacrolimus-related posterior reversible leukoencephalopathy (PRLE) is a rare complication which should be recognized by clinicians who regularly use immunosuppressive therapy. We report the case of an HIV-positive, hepatitis C-positive liver transplant patient who presented with this complication. Immunosuppression with tacrolimus was started after postsurgery. On the 20th day, the patient suffered two tonic-clonic convulsive attacks against a background of hypertension. Cerebral magnetic resonance imaging and lumbar puncture led to diagnosis of tacrolimus-related PRLE after eliminating other possible diagnoses. Therapeutic management consisted of withdrawing tacrolimus and initiating treatment with antiepileptogenic and antihypertensive drugs, supplemented with magnesium sulphate. The symptoms regressed in the days following withdrawal of tacrolimus and the majority of lesions on magnetic resonance imaging disappeared within two weeks. The aim of which should be to identify patients at risk of developing this complication. This would enable targeted prevention involving magnesium supplementation, strict control of blood pressure and serial monitoring of tacrolimus blood concentrations.

[Acute liver failure due to a treatment by nimesulide: another case and review]. / Hépatite fulminante liée à un traitement par nimésulide : encore un cas et revue de la littérature.

Nimesulide is a non-steroidal anti-inflammatory drug available in several European countries. A hepatic toxicity due to nimesulide has been reported but fatal cases remain rare. We report the case of a 49-year-old woman treated by nimesulide during three days, admitted to the intensive care unit for an acute liver failure with encephalopathy. A temporary hepatic support by molecular adsorbent recirculating system (MARS) was performed and a hepatic transplantation was performed 12 hours after admission, allowing a rapid improvement and a discharge from intensive care unit four days after transplantation. Nimesulide induced hepatic toxicity is unpredictable and the intensity of symptomatology is variable. Clinical symptoms are often progressive, delayed from the onset of treatment. Our case draws attention to the risk of hepatic failure related to treatment with nimesulide, leading to hepatic transplantation or death. The question of risk/benefit ratio must be asked again for this widely used molecule.

[Use of coupled plasma filtration adsorption for septic shock treatment]. / Utilisation de la coupled plasma filtration adsorption au cours du traitement d'un choc septique.

Coupled plasma filtration adsorption (CPFA) is a new technology proposed for septic shock, removing both pro- and anti-inflammatory mediators. We report the first use of CPFA in France. The patient was 80-years-old and was admitted to the intensive care unit because of septic shock from urinary source. Besides conventional septic shock treatment, four successive CPFA sessions were daily performed and lasted from 6 to 11 hours. At the end of the sessions, a noteworthy hemodynamic and inflammatory improvement was observed. This case confirms the feasibility and the relative simplicity of CPFA. It also illustrates the recent and few scientific data of the medical literature concerning this technology.

[Tracheal rupture after orotracheal intubation in intensive care]. / Rupture trachéale après intubation orotrachéale en réanimation.

We report a case of an iatrogenic tracheal rupture following an endotracheal intubation. The 78-year-old patient was admitted to the intensive care unit because of an acute respiratory failure related to a severe nosocomial pneumonia occurring 21 days after an abdominal aorta surgery. His main antecedent was a cigarette smoke-induced chronic obstructive pulmonary disease. Immediately after being intubated, a traumatic tracheobronchial rupture was suspected because of the sudden appearance of cervicothoracic subcutaneous emphysema. A thoracic computed tomography with multiplanar reformations confirmed the diagnosis and the evolution was unfortunately rapidly unfavourable. Risk factors, clinical and radiological aspects, and management of this rare but serious complication of endotracheal intubation will be discussed.

[Lung contusion: relevance of initial injured pulmonary volume measurement by computed tomography]. / Contusion pulmonaire: intérêt de l'évaluation du volume pulmonaire lésé en tomodensitométrie.

OBJECTIVE: To evaluate computed tomography quantification of injured pulmonary volume after thoracic trauma and its relevance for severity grade of patients with lung contusion. STUDY DESIGN: Retrospective study in a major French Level I university trauma center. PATIENTS AND METHODS: Clinical and biological data including oxygenation index (PaO2/FIO2) and therapeutics modalities during the first 5 days: positive end expiratory pressure (Peep) and nitric oxide (NO), were collected on 49 patients with lung contusion resulting from thoracic trauma. Injured pulmonary volume was evaluated on initial thoracic tomodensitometry by 2 senior radiologists. The correlation between oxygenation index, therapeutics modalities and initial injured pulmonary volume was assessed for signification. RESULTS: Injured pulmonary volume larger than 37.75% of total lung volume is associated with both hypoxemia at the twenty-fourth hour (PaO2/FIO2 <300), and need for Peep >6 cm H2O and /or ongoing NO administration on day 5. CONCLUSION: Injured parenchymal pulmonary volume evaluation on initial tomodensitometry seems to be an important indicator of lung contusion severity. Thoracic computed tomography provides additional prognostic information in the initial evaluation of thoracic trauma with parenchymal injury.

Granulocyte colony stimulating factors: how different are they? How to make a decision?

Two granulocyte colony stimulating factors (G-CSFs) are available for clinical use in Europe: filgrastim (Neupogen) and lenograstim (Granocyte). The purpose of this literature review is to study how they differ, the clinical implications of these differences (especially in terms of efficacy) and the economic impact of these differences. From a chemical point of view the two molecules are not identical. Their amino acid sequence is different and one is glycosylated, whereas the other is not. The important question to ask is what these structural differences mean for the patient. It appears that glycosylation has important consequences in terms of efficacy. Several recent comparative studies, both in vitro and in vivo, in animals and in humans, reinforce this idea which was often shared intuitively by physicians. In economical terms, in hospitals where the exact dosages are used (150 microg/m2 or 19.2 million units (MU)/m2 for Granocyte, and 5 microg/kg or 0.5 MU/kg for Neupogen), the choice of G-CSF must be made according to the daily cost of treatment which, for an average patient, means comparing the price of 325 microg of Neupogen and of 255 microg of Granocyte. This is in fact equal to comparing the price per MU of each product. In hospitals where one vial per patient per day is used whatever be their weight or body surface area, the price per MU and the price per vial should be considered together, puting into perspective the potential therapeutic benefit for patients, one vial of Granocyte 34 containing more MU than one vial of Neupogen 30.