Cécile Dubois

Bruno Vergès
email: bruno.verges@chu-dijon.fr

Rubrique Dossier thématique « Nouvelles perspectives en lipidologie » Sous rubrique Mise au point Les hypertriglycéridémies à l'origine de pancréatite aiguë Hypertriglyceridemias inducing acute pancreatitis

Keywords: Hypertriglycéridémie -Pancréatite aiguë -Étiologie --Traitement, ---------- Hypertriglyceridemia -Acute pancreatitis -Etiology -Treatment, ----------------

La pancréatite aiguë secondaire aux hypertriglycéridémies est en constante augmentation, et est vraisemblablement plus grave que les pancréatites aiguës d'autres origines. Il convient donc de connaître les caractéristiques spécifiques, ainsi que les facteurs de risque associés à cette pancréatite, afin de pouvoir la repérer précocement. Un diagnostic précis permet un traitement spécifique de l'épisode, avec parfois nécessité de recourir à la plasmaphérèse. Déterminer l'étiologie de l'hypertriglycéridémie est primordial afin de pouvoir la prendre en charge de façon optimale, et ainsi diminuer la survenue des pancréatites aiguës.

Introduction

La pancréatite aiguë (PA) induite par une hypertriglycéridémie (HTG) n'est pas si rare que ce que l'on pensait et est en augmentation croissante depuis plusieurs années. Elle représenterait 1 à 10 % de toutes les étiologies de PA, et même jusqu'à la moitié de tous les cas de PA pendant la grossesse. L'HTG est la cause de PA la plus fréquemment retrouvée après l'origine alcoolique et biliaire. Bien que la plupart des PA soient non graves, la mortalité peut atteindre jusqu'à 30 % [1]. L'hypertriglycéridémie (HTG) est définie par un taux de triglycéride plasmatique (TG) à jeun > 1,5 g/L. Selon le National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATP III), le taux de TG est à la limite haute entre 1,5 g/L et 1,99 g/L, est élevé entre 2 g/L et 4,99 g/L, et est très élevé si > 5 g/L. Les HTG sont des dyslipidémies appartenant à un ensemble très hétérogène, elles peuvent être primitive ou secondaire. L'étiologie de ces HTG est complexe car les causes primitives et secondaires sont souvent intriquées. Ces dernières années, devant l'amélioration du niveau de vie de la population générale, la morbidité induite par l'HTG n'a fait qu'augmenter, en particulier chez les jeunes personnes [2]. La prise en charge des HTG est donc un enjeu majeur, notamment lors des épisodes de PA.

Une physiopathologie encore floue

La physiopathologie de l'HTG induisant une PA n'est pas encore clairement élucidée. Des études sur des modèles animaux ont été réalisées, suggérant plusieurs théories qui ne sont pas exclusives : -la première théorie : l'HTG activerait l'hydrolyse par la lipase pancréatique de l'excès de triglycérides, ce qui formerait des acides gras libres. Ces acides gras libres formés à des concentrations élevées auraient une toxicité directe sur les cellules acinaires pancréatiques ; -la deuxième théorie : l'hyperviscosité secondaire à l'accumulation de chylomicrons dans les capillaires pancréatiques entraînerait une ischémie. Le fait que cette ischémie se produise uniquement dans le pancréas, et non dans les autres organes, reste une interrogation. Il existe également des caractéristiques génétiques spécifiques, une étude chinoise a mis en évidence une mutation du gène CFTR (pour Cystic Fibrosis Transmembrane Conductance Regulator) qui serait un facteur de risque indépendant de PA. En effet, dans cette étude, le taux de mutation du gène CFTR dans l'HTG avec PA était de 26,1 % (12 sur 46), alors qu'il n'était que de 1,3 % (1 sur 80) dans l'HTG sans PA [START_REF] Chang | Association of cystic fibrosis transmembrane conductance regulator (CFTR) mutation/variant/haplotype and tumor necrosis factor (TNF) promoter polymorphism in hyperlipidemic pancreatitis[END_REF]. Une étude espagnole a également montré que la mutation du gène ApoE était associée à la PA induite par une HTG [START_REF] Ivanova | Triglyceride levels and apolipoprotein E polymorphism in patients with acute pancreatitis[END_REF]. Il existe donc probablement une interaction entre toutes les théories décrites, avec une contribution variable selon les patients.

Comment reconnaitre une pancréatite aiguë secondaire à une

hypertriglycéridémie ? La PA induite par une HTG se présente cliniquement de la même façon que celles secondaires à d'autres étiologies. Le diagnostic peut être posé s'il existe deux des trois critères suivants : une douleur abdominale typique, une lipase sérique à plus de 3 fois la normale et/ou des images caractéristiques au scanner/TDM abdominal. Lors d'une HTG très sévère, certaines caractéristiques cliniques peuvent se voir, comme une xanthomatose éruptive, des xanthomes tubéreux, des xanthomes palmaires, ou encore une lipémie rétinienne.

La valeur seuil de TG pour affirmer l'étiologie de la PA n'est pas bien établie, mais généralement un taux de TG sérique > 10 g/L est retenu. En effet, cette valeur est arbitraire, et le niveau au-dessus duquel la PA peut se produire reste inconnu et varie selon les individus. Une étude prospective danoise indique que le risque de pancréatite aiguë est aussi augmenté pour des valeurs modérément élevées de TG, avec un risque relatif de 2,3 pour une triglycéridémie comprise entre 1,77 et 2,65 g/L, de 2,9 pour une triglycéridémie comprise entre 2,66 et 3,53 g/L, et de 3,9 pour une triglycéridémie comprise entre 3,54 et 4,42 g/L [START_REF] Pedersen | Non-fasting mild-to-moderate hypertriglyceridemia and risk of acute pancreatitis[END_REF]. À noter que les niveaux de TG ont tendance à diminuer rapidement pendant l'état de jeûne et le dosage des TG est souvent effectué à distance des premiers symptômes présentés par les patients, ce qui pourrait être susceptible d'entraîner une sous-évaluation des cas de PA liées à une HTG. L'HTG peut également être un épiphénomène accompagnant la PA. En effet, des valeurs de TG anormales sont présentes chez 50 % des patients ayant une PA quelle que soit l'étiologie, mais les taux de TG sont généralement < 10 g/L, et les troubles lipidiques se normalisent 72 h après le début des symptômes de la PA [START_REF] Linares | Acute pancreatitis in a cohort of 129 patients referred for severe hypertriglyceridemia[END_REF]. En pratique, il faut savoir que le sérum lactescent, rencontré dans les HTG sévères, peut perturber le dosage de l'amylase plasmatique qui, de ce fait, peut être anormalement « normal » [START_REF] Tremblay | Etiology and risk of lactescent plasma and severe hypertriglyceridemia[END_REF].

Identifier l'étiologie de l'hypertriglycéridémie : un enjeu majeur pour limiter le

risque de pancréatite aiguë L'HTG peut donc être primitive ou acquise, mais, généralement, elle résulte d'une combinaison de facteurs génétiques et environnementaux qu'il est nécessaire d'identifier pour limiter le risque de PA. Une étude japonaise composée de 215 patients atteints d'HTG > 10 g/L a suggéré que 74 % d'entre elles étaient induites par des causes secondaires [START_REF] Tada | Clinical characteristics of Japanese patients with severe hypertriglyceridemia[END_REF]. Les principales causes secondaires sont l'obésité, l'abus d'alcool, le diabète déséquilibré, l'hypothyroïdie non contrôlée, l'insuffisance hépatique et rénale grave, la grossesse, et l'utilisation de certaines thérapeutiques, comme les oestrogènes oraux, les diurétiques thiazidiques, les bêtabloquants, les corticostéroïdes, les rétinoïdes, les antipsychotiques atypiques, ou encore les agents antirétroviraux. Cependant, chez certains patients, l'HTG ne peut être contrôlée malgré la prise en charge des causes secondaires, et les caractéristiques génétiques peuvent alors être une explication. Actuellement, pour identifier une étiologie primaire, le séquençage des gènes LPL (Lipoprotéine lipase), LMF1 (Lipase maturation factor 1), GPIHBP1 (Glycosylphosphatidylinositol anchored High density lipoprotein Binding Protein 1), APOA5 (Apolipoprotein A5) et APOC2 (Apolipoprotein C2) est conseillé. Une étude française comprenant 129 sujets atteints d'HTG de type IV et V, a mis en évidence que 20,9 % d'entre eux ont connu au moins un épisode de PA. Dans cette même étude, un seul patient a obtenu un niveau de TG dans les normes après l'épisode de PA. Un niveau élevé de TG mesuré après un épisode de PA pourrait être considéré comme un marqueur d'HTG d'étiologie primaire [START_REF] Linares | Acute pancreatitis in a cohort of 129 patients referred for severe hypertriglyceridemia[END_REF].

Quels sont les facteurs de risque de développer une pancréatite aiguë en cas d'hypertriglycéridémie ?

Une étude ayant rassemblé 5550 sujets américains, a démontré que les patients présentant une HTG > 10 g/L avaient un risque plus élevé de développer une PA. En effet, 5,4 % des patients présentant une HTG très élevée ont développé une PA. De plus, il a été constaté que pour chaque augmentation de 1 g/L de TG au-dessus de 10 g/L, le risque de développer une PA augmente de 3 % [START_REF] Rashid | Severe hypertriglyceridemia and factors associated with acute pancreatitis in an integrated health care system[END_REF]. Dans l'étude française rassemblant des patients atteints d'hyperlipidémie de type IV et V, des niveaux moyens maximaux de TG plus élevé ont été constatés chez les patients ayant présenté une PA par rapport à ceux n'en n'ayant pas présenté [START_REF] Linares | Acute pancreatitis in a cohort of 129 patients referred for severe hypertriglyceridemia[END_REF]. Lors d'une étude chinoise composée de 149 personnes présentant une HTG sévère, une expansion du séquençage génétique comprenant 15 gènes liés aux triglycérides (LPL,

LMF1, APOC2, GPIHBP1, GCKR [Glucokinase regulator], ANGPTL3 [Angiopoietin like 3], APOB [Apolipoprotein E], APOA1-A4-C3-A5 [cluster Apolipoprotein A1-A4-C3-A5], TRIB1 [Tribbles pseudokinase 1], CETP [Cholesteryl ester transfer protein], APOE [Apolipoprotein E], et LIPI [Lipase 1
]) a été effectuée et a permis d'obtenir un taux de détection plus élevé de variantes rares. Il a été montré que des variants génétiques rares de la lipoprotéine lipase (LPL), de l'ApoA5, et de gènes régulant la LPL étaient associés à un risque accru de PA, chez les patients hypertriglycéridémiques [START_REF] Jin | Intensive genetic analysis for Chinese patients with very high triglyceride levels: Relations of mutations to triglyceride levels and acute pancreatitis[END_REF]. L'établissement d'un score génétique pourrait être un outil pour estimer le risque de PA chez les patients atteints d'HTG sévère. Dans une récente étude internationale rassemblant 1478 patients entre 2015 et 2018, il a été retrouvé que les patients ayant présenté une PA induite par une HTG (versus PA d'une autre étiologie) étaient, respectivement, plus jeunes (moyenne : 40 ans versus 50 ans), de sexe masculin (67 % versus 52 %) et avec un indice de masse corporelle (IMC) plus élevé (moyenne de 30,4 kg/m 2 versus 27,5 kg/m 2 ), De plus, la consommation d'alcool fréquente était plus importante (71 % versus 49 %), et il y avait un pourcentage plus élevé de patients diabétiques de type 2 (59 % versus 15 %) [START_REF] Pothoulakis | Clinical features of hypertriglyceridemia-induced acute pancreatitis in an international, multicenter, prospective cohort (APPRENTICE consortium)[END_REF]. Les antécédents de PA, l'insuffisance rénale sévère et l'hypertension artérielle, ont également été identifiés comme des facteurs de risque de PA lors d'HTG sévère, dans l'étude américaine citée précédemment. L'âge plus jeune et la consommation d'alcool étaient également retrouvés [START_REF] Rashid | Severe hypertriglyceridemia and factors associated with acute pancreatitis in an integrated health care system[END_REF]. Dans l'étude française, seul l'âge plus jeune était retenu comme facteur de risque de PA induite par une HTG [START_REF] Linares | Acute pancreatitis in a cohort of 129 patients referred for severe hypertriglyceridemia[END_REF].

Une sévérité controversée

Une étude a rassemblé 256 patients ayant souffert de PA secondaire à une HTG de 2016 à 2018. Celle-ci a mis en évidence que le taux sérique de TG au moment de l'admission était corrélé positivement avec la gravité de la PA [START_REF] Lu | Elevated triglycerides on admission positively correlate with the severity of hypertriglyceridaemic pancreatitis[END_REF]. Un taux élevé de TG était également positivement associé à un pronostic plus défavorable de PA dans une méta-analyse récente publiée en 2017, incluant 1564 patients ayant présenté une PA induite par une HTG et 5721 patients ayant souffert d'une PA d'une autre étiologie [2]. Dans une étude française, la gravité de la PA était supérieure à celle de la PA provoquée par une consommation d'alcool ou par celle liée à des calculs biliaires ; 71,5 % des patients ont présenté une PA grave avec une protéine C réactive (CRP) > 150 mg/L, un score de Balthazar supérieur à C, et une nécessité d'hospitalisation en soins intensifs [START_REF] Linares | Acute pancreatitis in a cohort of 129 patients referred for severe hypertriglyceridemia[END_REF]. Au contraire, dans une étude prospective récente, l'évolution clinique de ces sujets n'a pas été jugée plus compliquée que pour les patients atteints de PA d'autres étiologies [START_REF] Pothoulakis | Clinical features of hypertriglyceridemia-induced acute pancreatitis in an international, multicenter, prospective cohort (APPRENTICE consortium)[END_REF]. Cependant, dans la méta-analyse mentionnée ci-dessus, il a été mis en évidence une proportion plus importante d'insuffisance rénale, d'insuffisance respiratoire et de syndrome de réponse inflammatoire systémique (SRIS) lors des PA secondaires à une HTG par rapport aux PA d'autres causes. Il a également été objectivé un pronostic moins bon, avec une mortalité 1,9 fois plus élevée lors de PA dues à une HTG [2].

Traitements de l'hypertriglycéridémie en phase aiguë

La thérapie initiale de la PA induite par une HTG est la même que celle mise en place dans les pancréatites d'étiologies différentes. On distingue principalement trois axes : -la réhydratation intraveineuse ; -la nutrition adaptée ; -la prise en charge antalgique.

Les patients présentant une forme simple doivent bénéficier d'une réalimentation orale progressive après une période de jeûne qui peut durer plusieurs jours, et, lors des formes sévères, une nutrition artificielle est recommandée. L'objectif principal de la nutrition est d'aider l'organisme à faire face à l'hypermétabolisme et à l'hypercatabolisme, pour prévenir dans une certaine mesure la dénutrition [START_REF] Zazzo | Pancréatite aiguë : nutrition artificielle[END_REF]. Dans les situations d'HTG majeure avec une triglycéridémie > 10 g/L, les patients seront maintenus à jeun ou sous diète stricte sans aucun apport lipidique, sous contrôle métabolique en milieu hospitalier. Une prise en charge diététique plus précise sera initiée, dans un second temps, en milieu spécialisé, lorsque les valeurs de TG seront < 10 g/L et que le patient reprendra une alimentation per os. Des thérapeutiques favorisant la libération de la LPL sont disponibles, comme l'héparinothérapie et l'insulinothérapie. L'héparinothérapie permet le recrutement de la LPL sur la surface des cellules endothéliales, mais son effet est transitoire. Il faut cependant savoir que son action est peu efficace, voire nulle, en cas d'HTG sévère secondaire à une mutation homozygote de la LPL. L'insulinothérapie, quant à elle, mobilise la LPL synthétisée par les cellules musculaires et le tissu adipeux. À noter qu'il n'existe pas de recommandation d'expert pour l'utilisation de ces différentes thérapeutiques. Dans une étude publiée en 2018, un algorithme de la prise en charge spécifique de la PA secondaire à une HTG a été proposé. Tout d'abord, en parallèle des mesures communes, il est recommandé de mettre en place un traitement par insuline à un débit de 0,1 à 0,3 UI/kg/h pour abaisser le taux de TG, indépendamment de l'existence d'un diabète. Des contrôles glycémiques toutes les heures doivent être réalisés pour détecter d'éventuelles hypoglycémies. Si elles sont présentes, une solution de glucose intraveineux doit être ajoutée et, si l'hypoglycémie persiste, l'insuline doit être arrêtée. Les pancréatites sévères (score APACHE II > 7, score de Balthazar D-E, ou dysfonction d'au moins un organe) devraient être traitées de manière plus agressive en unité de soins intensifs à l'aide de traitement non médicamenteux, comme la plasmaphérèse [START_REF] Garg | Management of hypertriglyceridemia induced acute pancreatitis[END_REF]. Une étude sur l'utilisation de la plasmaphérèse lors des PA induites par une HTG a été publiée en 2019. Douze procédures ont été effectuées sur les huit patients de l'étude. Une réduction d'au moins 60 % du taux de TG a été obtenue dans 66,7 % de l'ensemble des procédures. Toutefois, le degré de réduction pour chaque procédure n'était pas prévisible [START_REF] Fei | Predictability and efficacy of therapeutic plasma exchange for hypertriglyceridemia induced acute pancreatitis[END_REF]. Si la plasmaphérèse n'est pas possible, des alternatives thérapeutiques existent, comme l'hémofiltration. Le traitement de réduction des TG doit être poursuivi jusqu'à ce que le taux soit < 5 g/L. De plus, il convient de commencer un traitement oral de réduction des lipides lorsque le patient est capable de tolérer une prise orale [START_REF] Garg | Management of hypertriglyceridemia induced acute pancreatitis[END_REF].

Traitements de l'hypertriglycéridémie en phase d'entretien

Comme signalé précédemment, le risque de développer une PA est plus important à mesure que le taux de TG augmente et s'il existe un antécédent de PA. Il convient donc de prendre en charge de façon optimale l'HTG après un épisode de PA secondaire à une HTG. Toutes les causes secondaires d'HTG sont à rechercher et sont à corriger. Les règles hygiéno-diététiques sont primordiales, elles consistent à réduire la charge calorique en cas de surpoids, de limiter l'apport des aliments à indice glycémique élevé, de consommer des poissons gras riches en oméga-3, d'obtenir un sevrage total et définitif de l'alcool, et de pratiquer une activité physique régulière. Les glycémies sont à normaliser lors d'un diabète déséquilibré, et les médicaments inducteurs d'HTG sont à arrêter dans la mesure du possible. Lors d'HTG sévères, les mesures hygiéno-diététiques peuvent ne pas suffire à atteindre un taux sérique de TG acceptable. L'adjonction d'un traitement médicamenteux est donc souvent nécessaire. Les fibrates en monothérapie sont à privilégier en 1 re intention lors d'HTG pures > 5 g/L malgré les mesures hygiéno-diététiques, tout en prenant en compte les différentes contre-indications et interactions médicamenteuses. S'il persiste une HTG > 5 g/L malgré les mesures hygiéno-diététiques et le traitement par fibrate à dose optimale, de l'acide gras oméga-3 à la dose de 4 g/j peut être ajouté. Les statines peuvent être utilisées lors d'HTG mixtes, mais leur effet est moindre sur l'HTG. L'association statine/fibrate peut également être utilisée, mais un avis spécialisé est préconisé [START_REF] Vergès | Prise en charge des dyslipidémies : quelles nouvelles recommandations ?[END_REF]. Lors des HTG primaires, il est nécessaire de réduire considérablement la charge lipidique, avec la mise en place de régimes extrêmement hypolipidiques (30 à 10 g de lipides par jour). L'utilisation de TG à chaîne moyenne (TCM) (NUTRICIA ® Huile de TCM, Nutricia Nutrition Clinique, Saint-Ouen) peut être intéressante dans ce contexte. Parfois, malgré la mise en place des différentes mesures citées ci-dessus, l'HTG persiste. De nouvelles molécules voient le jour. Un oligonucléotide antisens conçu pour inhiber la formation de l'ApoC-III (le volanesorsen, Waylivra ® , Akcea Therapeutics) est disponible depuis juillet 2019. Il est indiqué en complément d'un régime diététique chez les patients adultes atteints d'un syndrome d'hyperchylomicronémie familiale génétiquement confirmé et à risque élevé de PA, chez qui la réponse aux mesures diététiques et à un traitement de réduction des TG a été insuffisante [START_REF]Volanesorsen -WAYLIVRA 285 mg, solution injectable en seringue préremplie[END_REF].

Les points essentiels

• La pancréatite induite par l'hypertriglycéridémie (HTG) n'est pas exceptionnelle, elle représente jusqu'à 10 % de tous les épisodes de pancréatite aiguë (PA), et jusqu'à 50 % de tous les épisodes de PA pendant la grossesse. • Des taux sériques de triglycérides > 10 g/L sont jugés nécessaires pour établir la cause de la PA, mais ce seuil est arbitraire. • Il est essentiel d'identifier l'HTG comme cause de la PA grâce à un dosage précoce des triglycérides. • L'âge plus jeune est retrouvé lors des pancréatites secondaires à une HTG par rapport à celles d'autres étiologies. • La PA secondaire à une HTG semble plus sévère, et les complications semblent être corrélées au taux de triglycérides. • Le traitement initial est commun à celui utilisé pour les autres étiologies de PA, mais parfois, il est nécessaire d'adjoindre des moyens plus spécifiques, comme l'insulinothérapie ou encore la plasmaphérèse. • Déterminer l'étiologie de l'HTG est primordial pour pouvoir la prendre en charge de façon optimale, et ainsi diminuer le risque de survenue de PA.

Déclaration de liens d'intérêts

Les auteurs déclarent ne pas avoir de liens d'intérêts.

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  Références 1. Ewald N. Hypertriglyceridemia-induced acute pancreatitis. Clin Lipidol 2013; 8:587-94. 2. Wang Q, Wang G, Qiu Z, et al. Elevated serum triglycerides in the prognostic assessment of acute pancreatitis: a systematic review and meta-analysis of observational studies. J Clin Gastroenterol 2017; 51:586-93.


Maxime Fieux
email: maxime.fieux@chu-lyon.fr

Clémentine Daveau

Stéphane Tringali

Barotrauma and orbital pain

Case report

A female rugby player with no notable history (no history of trauma or surgery) consulted with a two-year history of painful episodes of very severe orbital congestion. The first episode occurred during a diving course while ascending to the surface from a depth of 18 metres. The patient experienced left eye pain with palpebral oedema that persisted for one week. This spontaneously resolving episode was followed by other very similar, but much less severe (sometimes simply consisting of blurred vision) and shorter episodes (several minutes to several hours). All episodes were triggered by pressure changes (at altitudes of more than 1,000 metres or while ascending from diving in a pool). Apart for these episodes, she was asymptomatic and was able to lead a normal life. She did not present any exophthalmos, conjunctival hyperaemia, or chemosis. Visual acuity, fundoscopy and optical coherence tomography of the optic disc were normal. Only visual field testing revealed two areas of decreased sensitivity in the left eye. Neurological examination was strictly normal. In this context of intermittent orbital symptoms with normal neuro-ophthalmological assessment, CT scan of the facial bones was performed (Figure 1).

Question

What is your diagnosis?

Answer

This patient presented pneumosinus dilatans of the left sphenoid sinus displacing the optic nerve without dehiscence of its bony canal. This case was presented to a skull base multidisciplinary consultation meeting, which validated surgical management. The operation was performed as day-case surgery under general anaesthesia after informing the patient about the benefits and risks (bleeding, infection, eyeball rupture and meningeal tear). The operation consisted of large sphenoidotomy to restore the communication between the sphenoid sinus and the nasal cavity in order to lower sinus pressure (Figure 2). Pneumosinus dilatans is an enlarged aerated paranasal sinus resulting in deformity of the adjacent bone, but without altering the bony walls of the sinus, unlike sinus pneumocele, as very well described by Prof. Bonfils [START_REF] Benkhatar | Mucocèles sinusiennes et pneumosinus dilatans[END_REF]. One hundred thirty-four cases of pneumosinus dilatans have been reported in the literature [START_REF] Ricci | Pneumosinus Dilatans: Over 100 Years Without an Etiology[END_REF]. The frontal sinus is the sinus most commonly involved (63% of cases), followed by the sphenoid sinus (24%), maxillary sinus (20%) and ethmoid sinus (19%). Surgery is only proposed in the presence of disabling aesthetic or functional impairment [START_REF] Benkhatar | Mucocèles sinusiennes et pneumosinus dilatans[END_REF]. The pathophysiology of pneumosinus dilatans remains unclear, but it is probably part of a continuum between hypersinus and pneumocele and would be secondary to progressive air trapping due to the presence of an anti-reflux valve effect in the sphenoethmoidal recess with progressively increasing sinus pressure [START_REF] Trimarchi | Pneumosinus dilatans of the maxillary sinus: a case report and review of the literature[END_REF]. Symptoms often occur during marked changes of atmospheric pressure, such as plane trips or underwater diving, as described in this patient [START_REF] Cervantes | Crista galli mucocele: endoscopic marsupialization via frontoethmoid approach[END_REF]. In contrast, the hypothesis of a spontaneously drained mucocele appears unlikely because of the absence of anterior or posterior rhinorrhoea. Sphenoid pneumosinus dilatans is classically asymptomatic, at least initially, or it may present in the form of visual field defects or loss of visual acuity, possibly resulting in blindness [START_REF] Desai | Pneumosinus dilatans: is it more than an aesthetic concern?[END_REF]. In view of this patient's unusual symptoms of intermittent pain with no loss of visual acuity or visual field defects on clinical examination, CT scan of the facial bones was not performed immediately. Although this disease has been well described in the literature, and although its pathophysiology remains controversial, the hypothesis of bone remodelling altering gas exchanges, thereby generating increased sinus pressure, is generally accepted. However, the clinical features associated with sphenoid pneumosinus dilatans in the form of severe orbital pain have never been reported, as pneumosinus dilatans of the frontal sinus is essentially responsible for pain in a context of barotrauma (plane trips or diving). Furthermore, this entity predominantly affects young men in their thirties and cases before the age of 20 years are exceptional [START_REF] Ricci | Pneumosinus Dilatans: Over 100 Years Without an Etiology[END_REF]. Imaging must be performed in patients reporting intermittent episodes of orbital pain associated with barotrauma. A normal neuro-ophthalmological examination can be falsely reassuring, leading to delayed radiological assessment.

The reference imaging modality is CT scan of the paranasal sinuses with 1 mm sections and 3D reconstruction. The imaging assessment has several objectives: i) establish a positive diagnosis; ii) detect any concomitant disease; iii) perform preoperative assessment looking for anatomical variants associated with a risk of complications, as treatment consists of endonasal surgery. This work-up can be completed by head MRI, as a concomitant disease is demonstrated in 50% of cases when pneumosinus dilatans involves only one sinus, and in 83% of cases when the

Figure 1 :

 1 Figure 1: CT scan of the paranasal sinuses, axial section, bone window setting.

Figure 2 :

 2 Figure 2: Intraoperative view of the left sphenoid sinus after sphenoidotomy.

sphenoid is involved [5]. Surgery consists of large sphenoidotomy designed to stop expansion of the paranasal sinus and decompress the optic nerve.

Conflicts of interest

The authors have no conflicts of interest to disclose.


MTD Y Lu

D C Wingate

Champion

R H Smithies

S P Johnson

K Gessner

R Maas

D R Mole

M Poujol

J Zhao

R A Creaser

Samarium-neodymium isotope map of Western Australia

. Here we present a revised Sm-Nd isotope map of Western Australia that updates previously released data with analyses of 230 new samples, for a total of 2224 analyses (Figs 1, 2).

These Sm-Nd isotope maps (Fig. 2) are based on whole-rock Sm-Nd data for felsic igneous rocks, which provide a window into the age and compositional variation of the middle and lower continental crust where most felsic magmas are generated [START_REF] Champion | Radiogenic isotopes, ore deposits and metallogenic terranes: Novel approaches based on regional isotopic maps and the mineral systems concept[END_REF]. Although mafic igneous and sedimentary rocks were not used in constructing the isotope maps, Sm-Nd data for those samples are also included in the data table along with data for felsic igneous rocks.

The isotope calculations are based on a chondritic uniform reservoir (CHUR) model with modern-day CHUR values of 147 Sm/ 144 Nd = 0.1960 and 143 Nd/ 144 Nd = 0.512630 [START_REF] Bouvier | The Lu-Hf and Sm-Nd isotopic composition of CHUR: constraints from unequilibrated chondrites and implications for the bulk composition of terrestrial planets[END_REF], a depleted mantle (DM) model with modern-day DM values of 147 Sm/ 144 Nd = 0.2136 and 143 Nd/ 144 Nd = 0.513163, and an assumed 147 Sm/ 144 Nd value of 0.11 for average continental crust [START_REF] Champion | Radiogenic isotopes, ore deposits and metallogenic terranes: Novel approaches based on regional isotopic maps and the mineral systems concept[END_REF]. The isotope maps were created using the Natural Neighbour interpolation tool in ArcGIS Spatial Analyst following [START_REF] Champion | Radiogenic isotopes, ore deposits and metallogenic terranes: Novel approaches based on regional isotopic maps and the mineral systems concept[END_REF], and are presented as both classified (natural breaks classification, Fig. 2a,c) and stretched (Histogram Equalize type, Fig. 2b,d) raster datasets.

The maps show two-stage depleted mantle model ages (TDM 2 , proxy for the average age of the crustal source of the igneous rocks) and crustal residence time (TCR, the difference between TDM 2 and magmatic crystallization age, i.e. the length of time the source of the igneous rocks has resided in the crust prior to melting). Strong isotope gradients are typically associated with major crustal boundaries and are potentially important for localizing mineral systems [START_REF] Martin | Major crustal boundaries map of Western Australia[END_REF]. However, map colours in areas with no or few samples reflect interpolated values and may have little or no relationship with underlying crust. Some isotope gradients may not be as pronounced in the statewide map as they might be on more detailed maps of individual regions. It is therefore recommended that users download the isotope data and create their own contour maps for particular areas, to enhance the isotope gradients in those areas. An example is provided for the Archean Yilgarn Craton (Fig. 3), which provides a more detailed image of the architecture of the Kalgoorlie-Kurnalpi region, a world-class Archean gold province [START_REF] Witt | The tectonic setting and evolution of the 2.7 Ga Kalgoorlie-Kurnalpi Rift, a world-class Archean gold province[END_REF]. It shows that old TDM 2 values (>2900 Ma) are common across the Yilgarn Craton, but only occur on the western and eastern margin of the Kurnalpi Terrane, supporting the interpretation that the Kalgoorlie-Kurnalpi region developed as a rift above Youanmi-like substrate with rifting centred in the Kurnalpi Terrane (e.g. [START_REF] Smithies | Two distinct origins for Archean greenstone belts[END_REF][START_REF] Mole | Time-space evolution of an Archean craton: A Hf-isotope window into continent formation[END_REF][START_REF] Witt | The tectonic setting and evolution of the 2.7 Ga Kalgoorlie-Kurnalpi Rift, a world-class Archean gold province[END_REF].

Insights into crustal evolution can be gained by visualizing the variation with age of Nd(i), TDM 2 and TCR for both mafic and felsic igneous rocks (Fig. 4). From c. 3730 to 2500 Ma, mafic and felsic igneous rocks show overlapping Nd(i), TDM 2 and TCR, which is in contrast to times younger than 2000 Ma, when mafic rocks generally yield higher Nd(i), and lower TDM 2 and TCR than felsic rocks in any given time. Felsic rocks older than 2500 Ma record sources requiring episodic juvenile mantle input at c. 3730, 3600, 3480-3420, 3300-3200, 3100, 2950, 2800 and 2700 Ma, as well as reworking of existing crust with TDM 2 up to c. 3960 Ma (Fig. 4a). The data for post-2000 Ma igneous rocks, however, suggest crustal reworking is the dominant mechanism forming felsic rocks younger than 2000 Ma in Western Australia. The increasing TCR for both mafic and felsic rocks from c. 3730 to 500 Ma is consistent with secular crustal maturation observed in other isotope systems, such as zircon oxygen isotopes (Fig. 4c; Valley et al., 2005;Lu et al., 2022).

There are few mafic or felsic igneous rocks between 2500 and 2000 Ma (Fig. 4). However, xenocrystic zircons of this age are common and have mainly heavy δ 18 O values, implying that felsic magmatism during this interval involved significant reworking of supracrustal materials, and that those magmatic rocks were mostly emplaced at depth and not exposed at the surface (Lu et al., 2022). This may indicate that the early Paleoproterozoic may have been dominated by orogenic compression, which trapped magmas at deep crustal levels [START_REF] Loucks | Deep entrapment of buoyant magmas by orogenic tectonic stress: Its role in producing continental crust, adakites, and porphyry copper deposits[END_REF]. The Sm-Nd isotope samples and associated data were compiled as part of a collaboration between GSWA and Geoscience Australia (GA). Acquisition of GSWA's Sm-Nd isotope data was funded by the Exploration Incentive Scheme (EIS), and involved collaboration with several university research laboratories.

How to access

The data layer is best accessed using GeoVIEW.WA. This online interactive mapping system allows data to be viewed and searched together with other datasets, including GSWA and GA geochronology data, geological maps, and mineral exploration datasets. The Samarium-neodymium isotope map digital data are also available as a free download from the Data and Software Centre via Datasets -Statewide spatial datasets -Geochronology & Isotope Geology -Samarium-neodymium isotope map, as ESRI shape files and MapInfo TAB files. These datasets are subject to ongoing updates as new data are generated.

Figure 1 .

 1 Figure 1. Locations of new and previously released Sm-Nd isotope samples, shown on the 1:2.5 million interpreted bedrock geology map

  Figure 2. Sm-Nd isotope maps for whole-rock samples of felsic igneous rocks in Western Australia. TDM 2 and TCR maps are presented as classified (a and c) and stretched (b and d) raster images. Symbols show the locations of Sm-Nd samples used for isotope mapping. Major crustal boundaries (gray lines) are from Martin et al. (2021)


Sandrine Pouvreau

Laszlo Csernoch

Bruno Allard

Jean Marc Sabatier

Michel De Waard

Michel Ronjat

Vincent Jacquemond
email: vincent.jacquemond@univ-lyon1.fr

TRANSIENT LOSS OF VOLTAGE CONTROL OF CA 2+ RELEASE IN THE PRESENCE OF MAUROCALCINE IN SKELETAL MUSCLE

Keywords: skeletal muscle, excitation-contraction coupling, intracellular calcium, ryanodine receptor

In skeletal muscle, sarcoplasmic reticulum (SR) calcium release is controlled by the plasma membrane voltage through interactions between the voltage sensing dihydropyridine receptor (DHPr) and the ryanodine receptor (RYr) calcium release channel. Maurocalcine (MCa), a scorpion toxin peptide presenting some homology with a segment of a cytoplasmic loop of the DHPr has been previously shown to strongly affect the activity of the isolated RYr. We injected MCa into mouse skeletal muscle fibers and measured intracellular calcium under voltage-clamp conditions. Voltage activated calcium transients exhibited similar properties in control and in MCa-injected fibers during the depolarizing pulses and the voltage dependence of calcium release was similar under the two conditions. However, MCa was responsible for a pronounced sustained phase of Ca 2+ elevation that proceeded for seconds following membrane repolarization, with no concurrent alteration of the membrane current. The magnitude of the underlying uncontrolled extra-phase of Ca 2+ release correlated well with the peak calcium release during the pulse. Results suggest that MCa binds to RYr that open upon membrane depolarization and that this interaction specifically alters the process of repolarization-induced closure of the channels.

INTRODUCTION

Optimal control of voluntary movement and force production relies ultimately on the efficacy of excitation-contraction (e-c) coupling of skeletal muscle. The contraction of a skeletal muscle fiber is rapidly initiated and stopped by changes in the voltage across the plasma membrane, physiologically an action potential or a train of action potentials. The coupling between membrane depolarization and the resulting transient rise in cytoplasmic [Ca 2+ ] that activates the contractile machinery is ensured by two types of ion channel proteins facing each other, across an ~15 nm wide gap at the triadic junction; these two are the DHPr in the plasma/transverse-tubule membrane, that senses the changes in voltage and the RYr calcium release channel in the SR membrane. The opening and closure of the RYr calcium channel is subordinated to voltage-dependent conformational changes of the DHPr but the details of the functional interactions between the two still remain obscure. Since the early nineties several lines of evidence have highlighted the importance of the II-III cytoplasmic loop of the α1 subunit of the DHPr in these interactions [START_REF] Tanabe | Regions of the skeletal muscle dihydropyridine receptor critical for excitation-contraction coupling[END_REF][START_REF] Lu | Activation of the skeletal muscle calcium release channel by a cytoplasmic loop of the dihydropyridine receptor[END_REF][START_REF] Nakai | Localization in the II-III loop of the dihydropyridine receptor of a sequence critical for excitation-contraction coupling[END_REF]. Within this loop, a 20 amino acid fragment named domain A has received particular attention because the corresponding isolated peptide is capable of modulating Ca 2+ release from SR preparations as well as the activity of the isolated RYr channel [START_REF] El-Hayek | Identification of calcium release-triggering and blocking regions of the II-III loop of the skeletal muscle dihydropyridine receptor[END_REF][START_REF] Dulhunty | Activation and inhibition of skeletal RyR channels by a part of the skeletal DHPR II-III loop: effects of DHPR Ser687 and FKBP12[END_REF][START_REF] Gurrola | Activation of ryanodine receptors by imperatoxin A and a peptide segment of the II-III loop of the dihydropyridine receptor[END_REF][START_REF] Casarotto | Structural determinants for activation or inhibition of ryanodine receptors by basic residues in the dihydropyridine receptor II-III loop[END_REF][START_REF] Stange | Two domains in dihydropyridine receptor activate the skeletal muscle Ca 2+ release channel[END_REF][START_REF] O'reilly | FKBP12 modulation of the binding of the skeletal ryanodine receptor onto the II-III loop of the dihydropyridine receptor[END_REF]. The situation is, however, not so clear since both activating and inhibiting effects have been reported. Furthermore the relevance and exact function of this segment during physiological e-c coupling has been seriously challenged by negative results in studies that used expression of chimeras of the α1 subunit in dysgenic myotubes [START_REF] Proenza | Excitation-contraction coupling is not affected by scrambled sequence in residues 681-690 of the dihydropyridine receptor II-III loop[END_REF][START_REF] Ahern | A component of excitation-contraction coupling triggered in the absence of the T671-L690 and L720-Q765 regions of the II-III loop of the dihydropyridine receptor α 1s pore subunit[END_REF][START_REF] Wilkens | Excitation-contraction coupling is unaffected by drastic alteration of the sequence surrounding residues L720-L764 of the α 1S II-III loop[END_REF].

MCa is a 33 amino acid toxin isolated from the venom of the scorpion Scorpio Maurus Palmatus [START_REF] Fajloun | Chemical synthesis and characterization of maurocalcine, a scorpion toxin that activates Ca 2+ release channel/ryanodine receptors[END_REF]. MCa is very similar to Imperatoxin A (IpTxa), a venom toxin from another scorpion and both share some sequence homology and a striking similarity in the spatial orientation of positively charged residues with domain A of the II-III loop of the DHPr (see [START_REF] Esteve | Critical amino acid residues determine the binding affinity and the Ca 2+ release efficacy of maurocalcine in skeletal muscle cells[END_REF][START_REF] Lee | Molecular basis of the high-affinity activation of type 1 ryanodine receptors by imperatoxin A[END_REF]. Interestingly both MCa and IpTxa can enhance [ 3 H] ryanodine binding to SR vesicles, increase ion flow through isolated ryanodine receptors, elicit Ca 2+ release from SR vesicles and modulate the properties of elementary calcium release events detected in permeabilized muscle fibers [START_REF] Gurrola | Activation of ryanodine receptors by imperatoxin A and a peptide segment of the II-III loop of the dihydropyridine receptor[END_REF][START_REF] Fajloun | Chemical synthesis and characterization of maurocalcine, a scorpion toxin that activates Ca 2+ release channel/ryanodine receptors[END_REF][START_REF] Esteve | Critical amino acid residues determine the binding affinity and the Ca 2+ release efficacy of maurocalcine in skeletal muscle cells[END_REF][START_REF] El-Hayek | Peptide probe of ryanodine receptor function. Imperatoxin A, a peptide from the venom of the scorpion Pandinus imperator, selectively activates skeletal-type ryanodine receptor isoforms[END_REF][START_REF] Tripathy | Imperatoxin A induces subconductance states in Ca 2+ release channels (ryanodine receptors) of cardiac and skeletal muscle[END_REF][START_REF] Shtifman | Effects of imperatoxin A on local sarcoplasmic reticulum Ca 2+ release in frog skeletal muscle[END_REF][START_REF] Szappanos | Differential effects of maurocalcine on Ca 2+ release events and depolarization-induced Ca 2+ release in rat skeletal muscle[END_REF]. In the case of MCa, there is also clear evidence that it binds to the same site of the RYr as domain A [START_REF] Altafaj | Maurocalcine and domain A of the II-III loop of the dihydropyridine receptor Cav 1.1 subunit share common binding sites on the skeletal ryanodine receptor[END_REF]. This potentially makes these two toxins highly interesting tools to characterize the still elusive role of domain A of the DHPr in the e-c coupling process. Here we injected MCa into adult skeletal muscle fibers from mouse and measured intracellular [Ca 2+ ] under voltage-clamp conditions. Results show that MCa dramatically affects the process of repolarization-induced closure of the RYrs during physiological e-c coupling.

METHODS

Preparation of the muscle fibers

Experiments were performed on single fibers isolated from the flexor digitorum brevis (fdb) muscles from 4-8 weeks-old Swiss OF1 mice. All experiments were performed in accordance with the guidelines of the French Ministry of Agriculture (87/848) and of the European Community (86/609/EEC). Procedures for enzymatic isolation of single muscle fibers, partial insulation of the fibers with silicone grease and intracellular micro-injection of the dye indo-1, were as described previously [START_REF] Jacquemond | Indo-1 fluorescence signals elicited by membrane depolarization in enzymatically isolated mouse skeletal muscle fibers[END_REF][START_REF] Collet | Intracellular calcium signals measured with indo-1 in isolated skeletal muscle fibres from control and mdx mice[END_REF][START_REF] Collet | Sustained release of calcium elicited by membrane depolarization in ryanodine-injected mouse skeletal muscle fibers[END_REF][START_REF] Collet | Calcium signaling in isolated skeletal muscle fibers investigated under "Silicone Voltage-Clamp" conditions[END_REF]. In brief, the major part of a single fiber was electrically insulated with silicone grease so that whole-cell voltage clamp could be achieved on a short portion of the fiber extremity. Prior to voltage-clamp, indo-1 was introduced into the myoplasm through local pressure micro-injection. Intracellular loading of either MCa or of its mutated analogue [Ala 24 ]MCa was achieved by co-injection with the dye; peptides were present in the micro-injected solution at a concentration of 100 µM. Following diffusion and equilibration within the cytoplasm, this was believed to achieve a final cytoplasmic concentration within the 10 micromolar range (for details concerning micro-injections see 25). It should be noted that the effects seen in the present study required two orders of magnitude more MCa than in experiments in more isolated systems. One likely reason for this discrepancy is that most of the injected MCa was bound to sites within the fiber so that the free MCa concentration was very much lower than the above estimated level.

In a separate series of experiments we also tested the effect of the injection of a synthetic domain A peptide corresponding to residues Thr 671 -Leu 690 of the α1 subunit of the DHPr; this peptide was used at a concentration of 2 mM in the injecting pipette. Control data were always obtained from fibers issued from the same muscles as the toxin-or peptide-injected fibers. The number of fibers tested under a given set of conditions was always obtained from muscles taken from at least 3 different animals.

All experiments were performed at room temperature (20-22 °C).

Electrophysiology

An RK-400 patch-clamp amplifier (Bio-Logic, Claix, France) was used in whole-cell configuration. Command voltage pulse generation and data acquisition were done using commercial software (Biopatch Acquire, Bio-Logic) driving an A/D, D/A converter (Lab Master DMA board, Scientific Solutions Inc., Mentor, OH, USA). Analog compensation was systematically used to decrease the effective series resistance. Voltage-clamp was performed with a microelectrode filled with the intracellular-like solution (see Solutions). The tip of the microelectrode was inserted through the silicone, within the insulated part of the fiber. Membrane depolarizations (or series of depolarizations) were applied every 30 s from a holding command potential of -80 mV.

Fluorescence measurements

The optical set-up and the procedures used for indo-1 fluorescence measurements were as described previously [START_REF] Jacquemond | Indo-1 fluorescence signals elicited by membrane depolarization in enzymatically isolated mouse skeletal muscle fibers[END_REF][START_REF] Collet | Intracellular calcium signals measured with indo-1 in isolated skeletal muscle fibres from control and mdx mice[END_REF][START_REF] Collet | Sustained release of calcium elicited by membrane depolarization in ryanodine-injected mouse skeletal muscle fibers[END_REF][START_REF] Pouvreau | Control of intracellular calcium in the presence of nitric oxide donors in isolated skeletal muscle fibres from mouse[END_REF]. In brief, a Nikon Diaphot epifluorescence microscope was used in diafluorescence mode. The beam of light from a high-pressure mercury bulb set on the top of the microscope was passed through a 335 nm interference filter and focused onto the preparation. The emitted indo-1 fluorescence light was collected by a 40x objective and simultaneously detected at 405 ± 5 nm (F 405 ) and 470 ± 5 nm (F 470 ) by two photomultipliers. The fluorescence measurement field was 40 µm in diameter and the silicone-free extremity of each tested fiber was placed in the middle of the field. Background fluorescence at both emission wavelengths was measured next to each tested fiber and was then subtracted from all measurements.

Calibration of the indo-1 response and [Ca 2+ ] i calculation

The standard ratio method was used with the parameters: R=F 405 /F 470 , and R min , R max , K D , β having their usual definition. Results were either expressed in terms of indo-1 percent saturation or in actual free calcium concentration (for details of calculation, see [START_REF] Jacquemond | Indo-1 fluorescence signals elicited by membrane depolarization in enzymatically isolated mouse skeletal muscle fibers[END_REF][START_REF] Csernoch | Measurements of intracellular Mg 2+ concentration in mouse skeletal muscle fibers with the fluorescent indicator mag-indo-1[END_REF]. In vivo values for R min , R max and β were measured using procedures described previously [START_REF] Collet | Intracellular calcium signals measured with indo-1 in isolated skeletal muscle fibres from control and mdx mice[END_REF][START_REF] Collet | Sustained release of calcium elicited by membrane depolarization in ryanodine-injected mouse skeletal muscle fibers[END_REF].

Calculation of the rate of calcium release

The method used to estimate the SR calcium release flux from the indo-1 transients was previously described [START_REF] Collet | Calcium signaling in isolated skeletal muscle fibers investigated under "Silicone Voltage-Clamp" conditions[END_REF]: the time derivative of the total myoplasmic Ca 2+ was calculated from the occupancy of the main intracellular calcium binding sites [START_REF] Baylor | Sarcoplasmic reticulum calcium release in frog skeletal muscle fibres estimated from Arsenazo III calcium transients[END_REF]. The model included troponin C binding sites with a total sites concentration TN total of 250 µM, an "on" rate constant k on, CaTN of 0.0575 µM -1 .ms -1 and an "off" rate constant k off, CaTN of 0.115 ms -1 ; Ca-Mg binding sites on parvalbumin with a total sites concentration PV total = 700 µM, "on" rate constant for Ca 2+ k on, CaPV = 0.125 µM -1 .ms -1 , "off" rate constant for Ca 2+ k off, CaPV = 5.10 -4 ms - 1 , "on" rate constant for Mg 2+ k on, MgPV = 3.3.10 -5 µM -1 .ms -1 , "off" rate constant for Mg 2+ k off, MgPV = 3.10 -3 ms -1 . The rate of calcium transport across the SR membrane was assumed to be proportional to the fractional occupancy of the SR pump sites with a dissociation constant Kd Ca pump of 2 µM and a maximum pump rate of 3 µM.ms -1 . Resting [Mg 2+ ] was assumed to be 1.5 mM. Indo-1 was assumed to be present at 100 µM with values for the "on" and "off" rate constants of the Ca-indo-1 binding reaction of 1.10 8 M -1 .s -1 and 30 s -1 , respectively.

Simulation of indo-1 transients

Simulations of [Ca-indo-1] transients elicited by membrane depolarizations were performed according to the procedure of Timmer et al. [START_REF] Timmer | Numerical methods to determine calcium release flux from calcium transients in muscle cells[END_REF], as described in Collet et al. [START_REF] Collet | Sustained release of calcium elicited by membrane depolarization in ryanodine-injected mouse skeletal muscle fibers[END_REF]. In brief, a synthetic Ca 2+ release rate waveform was fed into a calcium distribution model that reproduced the main features of Ca 2+ regulation in a muscle fiber. The model included the intracellular Ca 2+ binding sites and uptake mechanism described in the previous paragraph.

Resting [Ca 2+ ] was assumed to be 0.1 µM.

Solutions

The intracellular-like solution contained (in mM) 120 K-glutamate, 5 Na 2 -ATP, 5 Na 2phosphocreatine, 5.5 MgCl 2 , 5 glucose, 5 HEPES adjusted to pH 7.20 with K-OH. The standard extracellular solution contained (in mM) 140 TEA-methanesulfonate, 2.5 CaCl 2 , 2 MgCl 2 , 10 TEA-HEPES and 0.002 tetrodotoxin, pH 7.20.

Statistics

Least-squares fits were performed using a Marquardt-Levenberg algorithm routine included in Microcal Origin (Originlab, Northampton, MA, USA). Data values are presented as means ± S.E.M. for n fibers, where n is specified in Results. Statistical significance was determined using a Student's t-test assuming significance for P < 0.05.

RESULTS

The effect of MCa and of its mutated inactive analog [Ala 24 ]MCa [START_REF] Esteve | Critical amino acid residues determine the binding affinity and the Ca 2+ release efficacy of maurocalcine in skeletal muscle cells[END_REF] were tested on membrane current and intracellular calcium measured from isolated mouse skeletal muscle fibers. The mean initial resting [Ca 2+ ] level did not significantly differ between control fibers (58 ± 12 nM, n=16), MCa-injected fibers (49 ± 6 nM, n=22) and [Ala 24 ]MCa-injected fibers (62 ± 11 nM, n=7), indicating that the peptides did not chronically alter the intracellular resting Ca 2+ homeostasis. However, the presence of MCa produced a very remarkable change in the properties of the voltage activated Ca 2+ transients. This is illustrated in Fig. 1A which shows superimposed indo-1 calcium signals that were elicited by successive step membrane depolarizations of 10, 30 and 50 ms duration to +10 mV in a control fiber, an MCa-injected fiber and an [Ala 24 ]MCa-injected fiber. In all fibers, and as classically described, voltage activated SR Ca 2+ release produced a rapid rise in the calcium indicator saturation level and the signal remained elevated during the pulse. In the control fiber and in the [Ala 24 ]MCainjected fiber, membrane repolarization turned off Ca 2+ release and as a consequence the indo-1 signal returned towards its resting level with a time constant in the hundred of milliseconds range, as typically observed under normal conditions (see for instance [START_REF] Jacquemond | Indo-1 fluorescence signals elicited by membrane depolarization in enzymatically isolated mouse skeletal muscle fibers[END_REF][START_REF] Collet | Intracellular calcium signals measured with indo-1 in isolated skeletal muscle fibres from control and mdx mice[END_REF][START_REF] Pouvreau | Control of intracellular calcium in the presence of nitric oxide donors in isolated skeletal muscle fibres from mouse[END_REF]. In the MCa-injected fiber, there was also a repolarization-induced sharp decay of the indo-1 signal, but surprisingly the decline was only partial and the signal was still very much elevated at the end of the records. Pulses of 10 and 30 ms duration to + 10 mV were applied in several fibers issued from the same muscles upon these three conditions. The graphs in Fig. 1 give mean values for the corresponding baseline [Ca 2+ ] level (Fig. 1B), the peak ∆[Ca 2+ ] during the pulse (Fig. 1C), the ∆[Ca 2+ ] measured at the time of the end of the records shown in Fig. 1A (Fig. 1D, that is 0.92 s after the onset of the pulses, which we will refer to as 1 s after the pulses for simplicity) and the ratio of ∆[Ca 2+ ] measured 1 s after the pulse (∆[Ca 2+ ] 1s ) to the preceding peak ∆[Ca 2+ ] (Fig. 1E). Ca 2+ values were obtained without correction for the kinetics of indo-1 Ca-binding. Fibers were all challenged successively with 10 and 30 mslong pulses, with records taken either as shown in Fig. 1A or with longer records taken at a slower sampling rate (as shown in Fig. 2). There was no significant difference in the baseline [Ca 2+ ] (Fig. 1B) and peak ∆[Ca 2+ ] level (Fig. 1C) between the three groups of fibers. Conversely, the mean absolute ∆[Ca 2+ ] measured 1 s after the pulse was significantly larger, for both pulse duration, in the MCa-injected fibers than in either the control fibers or the [Ala 24 ]MCa-injected fibers (Fig. 1D). In average the ∆[Ca 2+ ] 1 s value was ~30 % of the preceding peak ∆[Ca 2+ ] in the MCa-injected fibers, and ~5 % in the control and in the [Ala 24 ]MCa-injected fibers (Fig. 1E). The fact that this ratio exhibited a similar value for the two pulses duration in the presence of MCa indicates that the amplitude of the phenomenon correlated well with the preceding extent of Ca 2+ release activation during the pulse.

In Fig. 1A, it is noticeable that within the 30 s period of time between two consecutive pulses, [Ca 2+ ] had eventually returned to its initial resting level as witnessed by the absence of change in the baseline indo-1 saturation level. The MCa-induced sustained post-pulse [Ca 2+ ] level was thus a transient phenomenon. Examples of its overall time course are illustrated in Fig. 2 which shows 15 s-long indo-1 saturation records in a control fiber and in two MCainjected fibers. Fibers were depolarized by short pulses to +10 mV of various duration. Although somewhat variable in pattern and amplitude, the post-pulse [Ca 2+ ] elevation observed in the presence of MCa always lasted for seconds and vanished with an extremely slow time course.

This long-lasting elevated intracellular [Ca 2+ ] level had to result from either an additional influx of Ca 2+ into-or a reduced Ca 2+ efflux from-the myoplasm. The possibility that MCa produced an extra-Ca 2+ influx across the plasma membrane following a step membrane depolarization is unlikely because there was no specific change in the membrane current during and after the pulses in the MCa-injected fibers, as compared to the control ones. This is illustrated in Fig. 3 which shows indo-1 calcium transients and corresponding membrane current traces displayed at a high y scale magnification, elicited by three consecutive 5 ms-long pulses to +10 mV applied to a control fiber (Fig. 3A) and to an MCainjected fiber (Fig. 3B). Whereas the presence of MCa induced a very reproducible longlasting Ca 2+ elevation after each of the pulses, the membrane current traces were very similar to those from the control fiber. Using data from the fibers used in Fig. 1, the mean change in membrane current ∆I (from the pre-pulse holding level) measured at the end of the 10 mslong pulses was -0.21 ± 0.3 (n=3), 0.72 ± 0.41 (n=5) and 1.46 ± 0.8 (n=5) A/F in control fibers, MCa-injected fibers and [Ala 24 ]MCa-injected fibers, respectively. When measured 1 s after the pulse, ∆I was -0.024 ± 0.006 (n=5), -0.021 ± 0.004 (n=9) and -0.01 ± 0.004 A/F (n=5), in the same groups, respectively. For the two sets of measurements there was no significant difference between the three groups.

Furthermore, in 3 fibers injected with MCa we took measurements in the presence and in the absence of extracellular calcium. For this each tested cell was superfused continuously with the extracellular solution using a polyethylene capillary perfusion system operated by gravity. Figure 4 shows membrane currents and indo-1 calcium signals measured from one of these fibers in response to a 20 ms long voltage step to +10 mV in the control condition (left), in the absence of extracellular calcium (replaced by magnesium, middle) and after returning to the control calcium containing solution (wash, right). The membrane current records are presented on an expanded x scale for clarity. They were corrected for the passive linear components by subtracting the adequately scaled current measured in response to a 20 mV hyperpolarization. The membrane current traces in the presence of the control external solution clearly exhibit a negative inward phase due to the activation of the slow inward calcium current (see for instance 30). As expected this current was lost in the absence of extracellular calcium but there was no simultaneous effect on the amplitude of the post-pulse sustained phase of [Ca 2+ ] elevation that resulted from the presence of MCa. In the 3 fibers tested the peak change in Ca 2+ measured 1 s after the end of the pulse in the absence of extracellular calcium corresponded to 86 ± 9 % of the value measured before in the presence of extracellular calcium. We believe that this definitely excludes the possibility of a significant contribution of external calcium to the effect of MCa.

The MCa-induced post-pulse elevated Ca 2+ level was thus of intracellular origin. Since the phenomenon was only triggered upon voltage activation of the fibers and because the initial fast decline of the calcium transients appeared unaffected, there is little doubt that the underlying mechanism was a transient dysfunction of SR Ca 2+ release. This of course is also in agreement with the results from previous measurements on SR vesicles and single ryanodine receptor channels that showed a selective effect of MCa on the calcium release channel activity with no concurrent alteration of the SERCA-mediated Ca 2+ uptake [START_REF] Esteve | Critical amino acid residues determine the binding affinity and the Ca 2+ release efficacy of maurocalcine in skeletal muscle cells[END_REF][START_REF] Chen | Maurocalcine and peptide A stabilize distinct subconductance states of ryanodine receptor type 1, revealing a proportional gating mechanism[END_REF]. The features of the MCa-induced post pulse Ca 2+ elevation are consistent with a slowly decaying extra-phase of Ca 2+ release proceeding for seconds following membrane repolarization. This is simulated in Fig. 5 using a model of calcium distribution (see Methods). Figure 5A shows synthetic Ca 2+ release flux traces in control conditions (thin trace) and in the presence of MCa (thick trace). The inset in Fig. 5A shows an enlarged view of the Ca 2+ release flux traces assumed to be activated by the depolarizing voltage step (V). The Ca 2+ flux waveform was made to have an early transient component that spontaneously decayed during the pulse towards a steady level. Upon repolarization, the flux was made to turn off with a time constant of 3 ms. Figure 5B shows the same traces on a longer time scale. The control and the MCa flux traces were made to be identical until the end of the pulse; thereafter the difference between the two was the presence of an additional small slowly decaying extra-phase of Ca 2+ release in the MCa trace. This slowly decaying component was simulated as having a single exponential time course with a time constant of 8 s and an amplitude of 0.2 µM/ms. Figure 5C and Fig. 5D show the indo-1 saturation traces calculated from the above fluxes on a short and a long time scale, respectively. Results clearly show that a slowly decaying post-pulse extra-Ca 2+ flux can well account for the results obtained in the presence of MCa. We also took advantage of the simulation to quantitatively estimate the amplitude of the transmembrane calcium current that would have been necessary to account for the observed effect of MCa (if the effect were to be solely due to Ca 2+ entry across the plasma membrane). Using a value of 40 µm for the diameter of the voltage-clamped end portion of the fiber and assuming a ratio of capacitance to apparent membrane surface of 5 µF/cm 2 (21, 30) and a fraction of total fiber volume accessible to Ca 2+ of 0.7, the MCainduced extra-Ca 2+ input flux of 0.2 µM/ms peak amplitude in Fig. 5 would have corresponded to a peak transmembrane calcium current of ~5 A/F amplitude. Since such a current was clearly absent from the records obtained in the presence of MCa (Fig. 3) this possibility was definitely discarded, in accordance with the results from Fig. 4.

As already pointed out in Fig. 1 and also observable in Fig. 2 a specific feature of the MCa effect was that the amplitude of the post-pulse [Ca 2+ ] elevation was larger as the pulse duration was increased, indicating that the size of the post-pulse uncontrolled Ca 2+ flux depended upon the previous extent of voltage-dependent Ca 2+ release activation. This was also true when, for a same duration, the pulse amplitude was increased within the voltage range of activation of Ca 2+ release (see Fig. 6A). This suggests that the more release channels are activated by voltage, the more will potentially be affected by MCa and remain open after membrane repolarization. Despite its impressive effect on the post-pulse [Ca 2+ ] level, MCa seemed to only marginally, if at all, affect Ca 2+ release during the depolarizing pulses. As an example, Fig. 6 shows the results from experiments aimed at comparing the voltage dependence of Ca 2+ release between control and MCa-injected fibers. Indo-1 Ca 2+ signals were recorded in response to the pulse protocol shown at the top of Fig. 6A. It consisted of a sequence of 3 consecutive step depolarizations of 30 ms duration to the indicated values; each pulse was then incremented by 5 mV and the sequence was repeated. Figure 6A shows an example of corresponding indo-1 saturation signals in a control fiber and in an MCa injected fiber while Fig. 6B shows the Ca 2+ release traces (d[Ca] T /dt, see Methods) calculated from the above transients. For clarity only the release flux trace calculated from the last sequence of indo-1 transients (in response to the pulses to -30, -10 and +10 mV) is shown. Although during a single sequence the sustained post-pulse [Ca 2+ ] level in the presence of MCa may have affected the properties of Ca 2+ release during the next pulse, the voltage level for both threshold and maximal activation of Ca 2+ release were similar in the two fibers. The mean voltage dependence of the peak Ca 2+ release flux from identical experiments in 3 control fibers and 4 MCa-injected fibers is shown in Fig. 6C. Superimposed curves were calculated with the mean parameters obtained from fitting a Boltzmann function to the individual series of data points in each fiber. Mean corresponding values for the peak release flux, midpoint voltage and steepness factor were 7.1 ± 0.6 µM/ms, -22 ± 2 mV and 4.6 ± 0.3 mV in control fibers and 5.4 ± 0.5 µM/ms, -17.3 ± 3 mV, 3.8 ± 0.1 mV in MCa-injected fibers, respectively. Mean values for the peak release flux and midpoint voltage did not significantly differ while the steepness factor was slightly but significantly reduced in the presence of MCa (P=0.05). Figure 6D presents the voltage dependence of the mean values for the post-pulse release flux in the control and in the MCa-injected fibers; values were measured 500 ms following the onset of each pulse (time indicated by the open arrows in panel B). In the MCa-injected fibers the amplitude of the post-pulse flux clearly had a voltage dependence similar to that of the peak release flux, whereas the control fibers exhibited no residual post-pulse flux. The strong dependence of the amplitude of the post-pulse release flux upon the amplitude of the peak flux is further illustrated in Fig. 6E where corresponding individual values for these two parameters were plotted versus each other. Fitting a straight line through the individual series of MCa data points in each fiber indicated that the post-pulse release flux corresponded to 4.3 ± 0.005 % of the peak flux activated during the pulse.

Since MCa presents some homology with domain A of the II-III loop of the α1 subunit of the DHPr, it was of particular interest to directly test the effect of domain A under the present conditions. A series of measurements was thus performed with fibers micro-injected with a synthetic domain A peptide (see Methods). Figure 7 illustrates a series of results from these experiments; this figure is organized in the same format as Figure 1 in order to ease the comparison. Figure 7A shows superimposed indo-1 calcium signals elicited by step depolarizations of 10, 20 and 50 ms duration to +10 mV in a control fiber an in a fiber injected with the domain A peptide. The graphs give mean values for the corresponding baseline [Ca 2+ ] level (Fig. 7B), the peak ∆[Ca 2+ ] during the pulse (Fig. 7C), the ∆[Ca 2+ ] measured 0.92 s (referred to as 1 s) after the onset of the pulses (Fig. 7D) and the ratio of ∆[Ca 2+ ] measured 1 s after the pulse (∆[Ca 2+ ] 1s ) to the preceding peak ∆[Ca 2+ ] (Fig. 7E). The y scale in panels B, D and E is the same as in the corresponding panels of Figure 1. Data are from 4 control fibers and 4 fibers injected with peptide A. Obviously, the presence of peptide A in the intracellular medium did not produce any reproducible alteration in the properties of the Ca 2+ transients that would be consistent with the effect of MCa.

DISCUSSION

The present results show that during physiological excitation-contraction coupling, the step of repolarization-induced closure of the Ca 2+ release channels is transiently disrupted by MCa, a toxin peptide that exhibits some homology with the domain A of the II-III loop of the DHPr in terms of both amino acid sequence (13) and three-dimensional structure [START_REF] Green | The three-dimensional structural surface of two β-sheet scorpion toxins mimics that of an α-helical dihydropyridine receptor segment[END_REF]. MCa was previously shown to have potentiating or activating effects on [ 3 H]ryanodine binding toand Ca 2+ release from-SR vesicles, on isolated RYr channel activity, on Ca 2+ release from intact myotubes and on spontaneous Ca 2+ release events in permeabilized muscle fibers [START_REF] Fajloun | Chemical synthesis and characterization of maurocalcine, a scorpion toxin that activates Ca 2+ release channel/ryanodine receptors[END_REF][START_REF] Esteve | Critical amino acid residues determine the binding affinity and the Ca 2+ release efficacy of maurocalcine in skeletal muscle cells[END_REF][START_REF] Szappanos | Differential effects of maurocalcine on Ca 2+ release events and depolarization-induced Ca 2+ release in rat skeletal muscle[END_REF][START_REF] Chen | Maurocalcine and peptide A stabilize distinct subconductance states of ryanodine receptor type 1, revealing a proportional gating mechanism[END_REF]. More recently MCa and domain A were shown to share common binding sites on RYr1 [START_REF] Altafaj | Maurocalcine and domain A of the II-III loop of the dihydropyridine receptor Cav 1.1 subunit share common binding sites on the skeletal ryanodine receptor[END_REF] which, added to the fact that MCa effect on RYr1 is clearly inhibited by domain A (29), would provide convincing support for the possibility that the two act on the same target through an analogous mechanism. The fact that the inhibition did not correspond to a simple competitive process ( 29) is likely to be due to the complexity of the overall mechanism because of the presumable 4:1 stoichiometry of the MCa (or domain A) -RYr1 interaction.

Here we show that, within the physiological operating mode of e-c coupling of an intact mammalian skeletal muscle fiber, MCa forces Ca 2+ release channels to remain open following a transient membrane depolarization. The effect of MCa was specific because a single amino-acid substitution (Arg 24 by an alanine), previously shown to prevent MCa alteration of RYr1 activity [START_REF] Esteve | Critical amino acid residues determine the binding affinity and the Ca 2+ release efficacy of maurocalcine in skeletal muscle cells[END_REF], induced the loss of its effectiveness. The simplest conclusion from our results is that repolarization-induced closure of MCa-modified RYr channels is temporarily impaired. At this point two alternatives may be considered.

The first possibility would be that the effects of MCa arise from its homology with domain A and tell us something about the dynamic physiological interactions that take place between the DHPr and the RYr during e-c coupling. In this framework however, one may have expected an excess of free peptide A in the myoplasm to produce a similar effect to that of MCa. In contrast, fibers injected with a synthetic domain A peptide did not exhibit a sustained Ca 2+ elevation following a step depolarization. This makes it hard to further argue in favor of this first hypothesis although it remains possible that the synthetic domain A peptide does not yield the exact native functional configuration of the corresponding endogenous segment within the II-III loop or that the exogenous domain A is functional but acts in an undistinguishable manner from the endogenous one. Alternatively, the difference observed here between peptide A and MCa in terms of functional effect may be related to the biochemical and single channel data indicating that peptide A does not compete with MCa [START_REF] Chen | Maurocalcine and peptide A stabilize distinct subconductance states of ryanodine receptor type 1, revealing a proportional gating mechanism[END_REF] or IpTx A (32) for certain effects on the ryanodine receptor. Finally, although here we did not detect any obvious effect of peptide A, it should be mentioned that in mechanically skinned rat skeletal muscle fibers with functional excitation-contraction coupling, peptide A was reported to elicit small spontaneous force responses in some fibers and to potentiate responses to depolarization in all fibers [START_REF] Lamb | Effect of dihydropyridine receptor II-III loop peptides on Ca 2+ release in skinned skeletal muscle fibers[END_REF].

Despite the sequence homology between domain A of the DHPr and MCa and their common binding site on the RYr, it is also possible that MCa acts through a mechanism that is independent from the function of domain A. Since the toxin had no detectable effect on resting [Ca 2+ ] in the absence of depolarization, MCa is likely to have poor access to its binding site under resting conditions. Depolarization-induced opening of the RYr channels most likely then reveals the MCa binding site, allowing interaction with the toxin. Ca 2+ transients exhibited no major alteration during the depolarizing pulses in the presence of MCa: the absolute peak amplitude of the transients and the voltage dependence of Ca 2+ release were similar to those observed under control conditions. MCa thus does not appear to strongly impair the functional operating mode of the overall population of RYr channels once the membrane is depolarized. This may indicate that MCa-bound channels behave normally during the depolarization though one also cannot exclude the possibility that the fraction of MCa-bound channels was low enough that even if operating in an altered mode their contribution would be too little to seriously affect the overall release flux during the pulse. Conversely, membrane repolarization clearly reveals the activity of the MCa-bound channels. Once bound, MCa thus likely prevents the conformational change required for channel closure upon membrane repolarization. In that sense MCa could be pictured to act in an analogous way as ryanodine which, as we have previously showed, produces a use-dependent increase in SR Ca 2+ leak due to RYr channels remaining locked open after a transient membrane depolarization [START_REF] Collet | Sustained release of calcium elicited by membrane depolarization in ryanodine-injected mouse skeletal muscle fibers[END_REF]. A noticeable difference is, however, that MCa-modified channels eventually return to a normal gating mode, presumably due to the dissociation of MCa, as indicated by the return of intracellular [Ca 2+ ] to its resting level.

The present effect of MCa has a striking similarity with a previously described alteration of e-c coupling in frog skeletal muscle where low myoplasmic [Mg 2+ ] levels could also induce a long-lasting elevation of myoplasmic [Ca 2+ ] following a transient membrane depolarization [START_REF] Jacquemond | Low myoplasmic Mg 2+ potentiates calcium release during depolarization of frog skeletal muscle fibers[END_REF]. The similarity with the present effect of MCa may be indicative that the putative site for Mg 2+ inhibition of Ca 2+ release is somehow linked to the MCa binding site.

In conclusion, this work demonstrates that the scorpion venom toxin MCa specifically alters the physiological process of repolarization-induced closure of the RYr channels. Further investigation of the properties of this effect will certainly prove helpful to unravel the molecular steps involved in the control of the RYr channels during excitation-contraction coupling. mV of various duration. Pulses of 5 and 10 ms duration were applied in the control fiber (left). Pulses of 5, 10 and 30 ms duration were applied in the two MCa-injected fibers.

Figure 3. The MCa-induced long-lasting post-pulse [Ca 2+

] elevation is not associated with a change in the holding membrane current. In A and B, the top series of traces correspond to successive (from left to right) indo-1 saturation transients elicited by a 5 ms long pulse to +10 mV in a control fiber (A) and in an MCa-injected fiber (B); the bottom series of traces show the corresponding membrane current records at a high gain. and in the presence of MCa (thick trace). In A and B the same traces are shown on a short and long time scale, respectively. In A, the inset focuses on the peak phase of the release traces. In B, the inset shows the two traces on an expanded y scale. The only difference between the control and the MCa trace is the presence of a small slowly decaying extra-phase of Ca 2+ release in the presence of MCa. This extra phase is best seen in the inset of panel B. C and D, corresponding calculated indo-1 saturation traces on a short and a long time scale, respectively. Figure 6. Voltage dependence of Ca 2+ release in control fibers and in MCainjected fibers. A, indo-1 saturation transients measured in response to the pulse protocol illustrated on top, in a control fiber (left) and in an MCa-injected fiber (right). The protocol consisted of a sequence of 3 consecutive step depolarizations of 30 ms duration. During the first sequence the 3 successive steps were to -50, -30 and -10 mV, respectively. The amplitude of each of the 3 steps was then incremented by 5 mV and the sequence was repeated. The sequence was applied 5 times so that during the last run the 3 steps were to -30, -10 and +10 mV, respectively. Indo-1 transients obtained in response to the 5 sequences are shown superimposed. B, Ca 2+ release flux calculated from the above indo-1 transients; only the release flux from the indo-1 trace obtained in response to the successive pulses to -30, -10 and +10 mV is shown. The y scale bar in each panel corresponds to 1 µM/ms. The horizontal dotted line corresponds to the initial baseline level. Filled and open arrows point to the peak and post-pulse release flux levels that were measured, respectively; amplitudes were measured from the pre-pulse level. The post-pulse flux values were taken as the average from a 100 ms long portion of trace, 500 ms following the pulse onset. The mean from these measurements is reported in the following panels. C, mean voltage dependence of the peak Ca

Figure 1 .

 1 Figure 1. Incomplete decay of step depolarization-activated Ca 2+ transients in MCa-injected fibers. A, indo-1 saturation transients elicited by consecutive depolarizing steps of 10, 30 and 50 ms duration to +10 mV in a control fiber (top) in an MCa-injected fiber (middle) and in a fiber injected with the mutated analog [Ala 24 ]MCa. B, mean values for the baseline [Ca 2+ ] level in control (n=5), MCa-injected (n=9) and [Ala 24 ]MCa-injected fibers (n=5) challenged by a 10 and a 30 ms-long pulse to +10 mV. C and D, corresponding values for the peak ∆[Ca 2+ ] and the ∆[Ca 2+ ] measured ~1 s after the onset of the pulse, respectively. E, corresponding mean values for the ratio of the ∆[Ca 2+ ] measured ~1 s after the onset of the pulse (mean shown in C) to the peak ∆[Ca 2+ ] during the pulse (mean shown in D). See text for details.

Figure 2 .

 2 Figure 2. The long lasting [Ca 2+ ] elevation following the end of a step depolarization in MCa-injected fibers. Traces correspond to indo-1 saturation records of 15 s duration in a control fiber and in two MCa-injected fibers depolarized by short pulses to +10

Figure 4 .

 4 Figure 4. The MCa-induced long-lasting post-pulse [Ca 2+ ] elevation persists in the absence of extracellular calcium. All records are from the same fiber injected with MCa. The top series of traces corresponds to the changes in membrane current elicited by a 20 ms long pulse to +10 mV. The indo-1 calcium signals measured simultaneously are shown underneath on a more compressed time scale. Records were taken in the presence of the control extracellular solution (left), in the absence of extracellular calcium (middle) and after returning to the calcium containing control solution (right). Membrane current records were corrected for the linear components.

Figure 5 .

 5 Figure 5. Simulation of the effect of MCa using a simple model of intracellular Ca 2+ distribution. A and B, synthetic Ca 2+ release flux traces in control conditions (thin trace) and in the presence of MCa (thick trace). In A and B the same traces are shown on a short and long time scale, respectively. In A, the inset focuses on the peak phase of the release traces. In B, the inset shows the two traces on an expanded y scale. The only difference between the control and the MCa trace is the presence of a small slowly decaying extra-phase of Ca 2+ release in the presence of MCa. This extra phase is best seen in the inset of panel B. C and D, corresponding calculated indo-1 saturation traces on a short and a long time scale, respectively. Figure6. Voltage dependence of Ca 2+ release in control fibers and in MCainjected fibers. A, indo-1 saturation transients measured in response to the pulse protocol illustrated on top, in a control fiber (left) and in an MCa-injected fiber (right). The protocol consisted of a sequence of 3 consecutive step depolarizations of 30 ms duration. During the first sequence the 3 successive steps were to -50, -30 and -10 mV, respectively. The amplitude of each of the 3 steps was then incremented by 5 mV and the sequence was repeated. The sequence was applied 5 times so that during the last run the 3 steps were to -30, -10 and +10 mV, respectively. Indo-1 transients obtained in response to the 5 sequences are shown superimposed. B, Ca 2+ release flux calculated from the above indo-1 transients; only the release flux from the indo-1 trace obtained in response to the successive pulses to -30, -10 and +10 mV is shown. The y scale bar in each panel corresponds to 1 µM/ms. The horizontal dotted line corresponds to the initial baseline level. Filled and open arrows point to the peak and post-pulse release flux levels that were measured, respectively; amplitudes were measured from the pre-pulse level. The post-pulse flux values were taken as the average from a 100 ms long portion of trace, 500 ms following the pulse onset. The mean from these measurements is reported in the following panels. C, mean voltage dependence of the peak Ca 2+ release flux in control fibers (filled circles, n=3) and in MCa-injected fibers (open circles, n=4). D, corresponding mean voltage dependence of the post-pulse Ca 2+ release flux. E, plot of the individual values of the post-pulse release flux versus the corresponding peak release flux in the control (filled symbols) and the MCa-injected fibers (open symbols). Different symbols correspond to the different fibers.

  Figure 5. Simulation of the effect of MCa using a simple model of intracellular Ca 2+ distribution. A and B, synthetic Ca 2+ release flux traces in control conditions (thin trace) and in the presence of MCa (thick trace). In A and B the same traces are shown on a short and long time scale, respectively. In A, the inset focuses on the peak phase of the release traces. In B, the inset shows the two traces on an expanded y scale. The only difference between the control and the MCa trace is the presence of a small slowly decaying extra-phase of Ca 2+ release in the presence of MCa. This extra phase is best seen in the inset of panel B. C and D, corresponding calculated indo-1 saturation traces on a short and a long time scale, respectively. Figure6. Voltage dependence of Ca 2+ release in control fibers and in MCainjected fibers. A, indo-1 saturation transients measured in response to the pulse protocol illustrated on top, in a control fiber (left) and in an MCa-injected fiber (right). The protocol consisted of a sequence of 3 consecutive step depolarizations of 30 ms duration. During the first sequence the 3 successive steps were to -50, -30 and -10 mV, respectively. The amplitude of each of the 3 steps was then incremented by 5 mV and the sequence was repeated. The sequence was applied 5 times so that during the last run the 3 steps were to -30, -10 and +10 mV, respectively. Indo-1 transients obtained in response to the 5 sequences are shown superimposed. B, Ca 2+ release flux calculated from the above indo-1 transients; only the release flux from the indo-1 trace obtained in response to the successive pulses to -30, -10 and +10 mV is shown. The y scale bar in each panel corresponds to 1 µM/ms. The horizontal dotted line corresponds to the initial baseline level. Filled and open arrows point to the peak and post-pulse release flux levels that were measured, respectively; amplitudes were measured from the pre-pulse level. The post-pulse flux values were taken as the average from a 100 ms long portion of trace, 500 ms following the pulse onset. The mean from these measurements is reported in the following panels. C, mean voltage dependence of the peak Ca 2+ release flux in control fibers (filled circles, n=3) and in MCa-injected fibers (open circles, n=4). D, corresponding mean voltage dependence of the post-pulse Ca 2+ release flux. E, plot of the individual values of the post-pulse release flux versus the corresponding peak release flux in the control (filled symbols) and the MCa-injected fibers (open symbols). Different symbols correspond to the different fibers.

Figure 7 .

 7 Figure 7. Voltage-activated Ca 2+ transients in fibers injected with a synthetic peptide corresponding to domain A of the II-III loop of the DHPr α α α α1 subunit. A, indo-1 saturation transients elicited by consecutive depolarizing steps of 10, 20 and 50 ms duration to

  Figure 4

ACKNOWLEDGEMENTS

This work was supported by grants from CNRS, University Claude Bernard Lyon 1 and Association Française contre les Myopathies.