Vascular smooth muscle cell phenotypic modulation in culture is associated with reorganisation of contractile and cytoskeletal proteins

Smooth muscle cells (SMC) exhibit a functional plasticity, modulating from the mature phenotype in which the primary function is contraction, to a less differentiated state with increased capacities for motility, protein synthesis, and proliferation. The present study determined, using Western analysis, double-label immunofluorescence and confocal microscopy, whether changes in phenotypic expression of rabbit aortic SMC in culture could be correlated with alterations in expression and distribution of structural proteins. Contractile state SMC (days 1 and 3 of primary culture) showed distinct sorting of proteins into subcellular domains, consistent with the theory that the SMC structural machinery is compartmentalised within the cell. Proteins specialised for contraction (alpha -SM actin, SM-MHC, and calponin) were highly expressed in these cells and concentrated in the upper central region of the cell. Vimentin was confined to the body of the cell, providing support for the contractile apparatus but not co-localising with it. In line with its role in cell attachment and motility, beta -NM actin was localised to the cell periphery and basal cortex. The dense body protein alpha -actinin was concentrated at the cell periphery, possibly stabilising both contractile and motile apparatus. Vinculin-containing focal adhesions were well developed, indicating the cells' strong adhesion to substrate. In synthetic state SMC (passages 2-3 of culture), there was decreased expression of contractile and adhesion (vinculin) proteins with a concomitant increase in cytoskeletal proteins (beta -non-muscle [NM] actin and vimentin). These quantitative changes in structural proteins were associated with dramatic chan-es in their distribution. The distinct compartmentalisation of structural proteins observed in contractile state SMC was no longer obvious, with proteins more evenly distributed throughout die cytoplasm to accommodate altered cell function. Thus, SMC phenotypic modulation involves not only quantitative changes in contractile and cytoskeletal proteins, but also reorganisation of these proteins. Since the cytoskeleton acts as a spatial regulator of intracellular signalling, reorganisation of the cytoskeleton may lead to realignment of signalling molecules, which, in turn, may mediate the changes in function associated with SMC phenotypic modulation. (C) 2001 Wiley-Liss, Inc.

Effects of vitamin E deficiency on fatigue and muscle contractile properties

Radical-mediated oxidative damage of skeletal muscle membranes has been implicated in the fatigue process. Vitamin E (VE) is a major chain breaking antioxidant that has been shown to reduce contraction-mediated oxidative damage. We hypothesized that VE deficiency would adversely affect Muscle contractile function, resulting in a more rapid development of muscular fatigue during exercise. To test this postulate, rats were fed either a VE-deficient (EDEF) diet or a control (CON) diet containing VE. Following a 12-week feeding period, animals were anesthetized and mechanically ventilated. Muscle endurance (fatigue) and contractile properties were evaluated using an in situ preparation of the tibialis anterior (TA) muscle. Contractile properties of the TA muscle were determined before and after a fatigue protocol. The muscle fatigue protocol consisted of 60 min of repetitive contractions (250 ms trains at 15 Hz; duty cycle = I I %) of the TA muscle. Prior to the fatigue protocol, no significant differences existed in the force-frequency curves between EDEF and CON animals. At the completion of the fatigue protocol, muscular force production was significantly (P

Myoelectric manifestations of sternocleidomastoid and anterior scalene muscle fatigue in chronic neck pain patients

Objective: This study compares myoelectric manifestations of fatigue of the sternocleidomastoid (SCM) and anterior scalene (AS) muscles between 10 chronic neck pain subjects and 10 normal matched controls. Methods: Surface electromyography (sEMG) signals were recorded from the sternal bead of SCM and AS muscles bilaterally during submaximal isometric cervical flexion contractions at 25 and 50% of the maximum voluntary contraction (MVC). The mean frequency, average rectified value and conduction velocity of the sEMG signal were calculated to quantify myoelectric manifestations of muscle fatigue. Results: For both the SCM and AS muscles, the Mann-Whitney U test indicated that the initial value and slope of the mean frequency in neck pain patients were greater than in healthy subjects (P < 0.05). This was significant both at 25 and 50% of MVC. Conclusions: These results suggest: (a) a predominance of type-II fibres in the neck pain patients and/or (b) greater fatigability of the superficial cervical flexors in neck pain patients. These results are in agreement with previous muscle biopsy studies in subjects with neck pain, which identified transformation of slow-twitch type-I fibres to fast-twitch type-IIB fibres, as well as the clinical observation of reduced endurance in the cervical flexors in neck pain patients. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.

Spatiotemporal mechanisms for actomyosin ring assembly and contraction in budding yeast cell division

Dissertation presented to obtain the Ph.D degree in Molecular Medicine

Effects of protein-calorie restriction on mechanical function of hypertrophied cardiac muscle

OBJECTIVE: To assess the effect of food restriction (FR) on hypertrophied cardiac muscle in spontaneously hypertensive rats (SHR). METHODS: Isolated papillary muscle preparations of the left ventricle (LV) of 60-day-old SHR and of normotensive Wistar-Kyoto (WKY) rats were studied. The rats were fed either an unrestricted diet or FR diet (50% of the intake of the control diet) for 30 days. The mechanical function of the muscles was evaluated through monitoring isometric and isotonic contractions. RESULTS: FR caused: 1) reduction in the body weight and LV weight of SHR and WKY rats; 2) increase in the time to peak shortening and the time to peak developed tension (DT) in the hypertrophied myocardium of the SHR; 3) diverging changes in the mechanical function of the normal cardiac muscles of WKY rats with reduction in maximum velocity of isotonic shortening and of the time for DT to decrease 50% of its maximum value, and increase of the resting tension and of the rate of tension decline. CONCLUSION: Short-term FR causes prolongation of the contraction time of hypertrophied muscles and paradoxal changes in mechanical performance of normal cardiac fibers, with worsening of the shortening indices and of the resting tension, and improvement of the isometric relaxation.

Biometal muscle to restore atrial transport function in a permanent atrial fibrillation animal model: a potential tool in the treatment of end-stage heart failure.

BACKGROUND: Half of the patients with end-stage heart failure suffer from persistent atrial fibrillation (AF). Atrial kick (AK) accounts for 10-15% of the ejection fraction. A device restoring AK should significantly improve cardiac output (CO) and possibly delay ventricular assist device (VAD) implantation. This study has been designed to assess the mechanical effects of a motorless pump on the right chambers of the heart in an animal model. METHODS: Atripump is a dome-shaped biometal actuator electrically driven by a pacemaker-like control unit. In eight sheep, the device was sutured onto the right atrium (RA). AF was simulated with rapid atrial pacing. RA ejection fraction (EF) was assessed with intracardiac ultrasound (ICUS) in baseline, AF and assisted-AF status. In two animals, the pump was left in place for 4 weeks and then explanted. Histology examination was carried out. The mean values for single measurement per animal with +/-SD were analysed. RESULTS: The contraction rate of the device was 60 per min. RA EF was 41% in baseline, 7% in AF and 21% in assisted-AF conditions. CO was 7+/-0.5 l min(-1) in baseline, 6.2+/-0.5 l min(-1) in AF and 6.7+/-0.5 l min(-1) in assisted-AF status (p<0.01). Histology of the atrium in the chronic group showed chronic tissue inflammation and no sign of tissue necrosis. CONCLUSIONS: The artificial muscle restores the AK and improves CO. In patients with end-stage cardiac failure and permanent AF, if implanted on both sides, it would improve CO and possibly delay or even avoid complex surgical treatment such as VAD implantation.

Artificial muscle to wash blood out of fibrillating atrium: an alternative to lifelong anticoagulation.

The Atripump is a motorless, volume displacement pump based on artificial muscle technology that could reproduce the pump function of normal atrium. It could help prevent blood clots due to blood stagnation and eventually avoid anticoagulation therapy in atrial fibrillation (AF). An animal study has been designed to assess mechanical effects of this pump on fibrillating atrium. The Atripump is a dome shaped silicone coated nitinol actuator. A pacemaker like control unit drives the actuator. In five adult sheep, the right atrium (RA) was exposed and dome sutured onto the epicardium. Atrial fibrillation was induced using rapid epicardial pacing (600 beats/min). Ejection fraction of the RA was obtained with intracardiac ultrasound in baseline, AF and Atripump assisted AF conditions. The dome's contraction rate was 60/min with power supply of 12V, 400 mA for 200 ms and ran for 2 hours in total. Mean temperature on the RA was 39+/-1.5 degrees C. Right atrium ejection fraction was 31% in baseline conditions, 5% and 20% in AF and assisted AF, respectively. In two animals a thrombus appeared in the right appendix and washed out once the pump was turned on. The Atripump washes blood out the RA acting as an anticoagulant device. Possible clinical implications in patients with chronic AF are prevention of embolism of cardiac origin and avoidance of hemorrhagic complication due to chronic anticoagulation.

Techniques d'évaluation de la force des muscles respiratoires. [Techniques for assessing respiratory muscle strength.]

La faiblesse des muscles respiratoires peut entraîner une dyspnée, un encombrement bronchique et une insuffisance respiratoire potentiellement fatale. L'évaluation de la force musculaire respiratoire s'impose donc dans les affections neuro-musculaires, mais également dans les situations de dyspnée inexpliquée par une première évaluation cardiaque et pulmonaire. À la spirométrie, une faiblesse musculaire est suspectée sur la base de la boucle débit-volume montrant un débit de pointe émoussé et une fin prématurée de l'expiration. Une diminution importante de la capacité vitale en position couchée suggère une paralysie diaphragmatique. La force inspiratoire est mesurée par la pression inspiratoire maximale (PImax) contre une quasi-occlusion des voies aériennes. Ce test relativement difficile est d'interprétation délicate en cas de collaboration insuffisante. La mesure de la pression nasale sniff (SNIP) est une alternative utile, car elle élimine le problème des fuites autour de l'embout buccal et la réalisation du reniflement est facile. De même, la pression trans-diaphragmatique sniff mesure la force du diaphragme au moyen de sondes oesophagienne et gastrique. En cas de collaboration insuffisante, on peut recourir à la stimulation magnétique des nerfs phréniques qui induit une contraction non-volontaire du diaphragme. La force expiratoire est mesurée par la pression expiratoire maximale (PEmax) contre une quasi-occlusion. La force disponible pour tousser est mesurée par la pression gastrique à la toux, ou plus simplement par le débit de pointe à la toux. Chez les patients à risque, la mesure de la force des muscles respiratoires permet d'instaurer à temps une assistance ventilatoire ou à la toux.

Wide-pulse-high-frequency neuromuscular stimulation of triceps surae induces greater muscle fatigue compared with conventional stimulation.

We compared the extent and origin of muscle fatigue induced by short-pulse-low-frequency [conventional (CONV)] and wide-pulse-high-frequency (WPHF) neuromuscular electrical stimulation. We expected CONV contractions to mainly originate from depolarization of axonal terminal branches (spatially determined muscle fiber recruitment) and WPHF contractions to be partly produced via a central pathway (motor unit recruitment according to size principle). Greater neuromuscular fatigue was, therefore, expected following CONV compared with WPHF. Fourteen healthy subjects underwent 20 WPHF (1 ms-100 Hz) and CONV (50 μs-25 Hz) evoked isometric triceps surae contractions (work/rest periods 20:40 s) at an initial target of 10% of maximal voluntary contraction (MVC) force. Force-time integral of the 20 evoked contractions (FTI) was used as main index of muscle fatigue; MVC force loss was also quantified. Central and peripheral fatigue were assessed by voluntary activation level and paired stimulation amplitudes, respectively. FTI in WPHF was significantly lower than in CONV (21,717 ± 11,541 vs. 37,958 ± 9,898 N·s P<0,001). The reductions in MVC force (WPHF: -7.0 ± 2.7%; CONV: -6.2 ± 2.5%; P < 0.01) and paired stimulation amplitude (WPHF: -8.0 ± 4.0%; CONV: -7.4 ± 6.1%; P < 0.001) were similar between conditions, whereas no change was observed for voluntary activation level (P > 0.05). Overall, our results showed a different motor unit recruitment pattern between the two neuromuscular electrical stimulation modalities with a lower FTI indicating greater muscle fatigue for WPHF, possibly limiting the presumed benefits for rehabilitation programs.

Intercellular calcium waves in primary cultured rat mesenteric smooth muscle cells are mediated by connexin43.

Intercellular Ca(2+) wave propagation between vascular smooth muscle cells (SMCs) is associated with the propagation of contraction along the vessel. Here, we characterize the involvement of gap junctions (GJs) in Ca(2+) wave propagation between SMCs at the cellular level. Gap junctional communication was assessed by the propagation of intercellular Ca(2+) waves and the transfer of Lucifer Yellow in A7r5 cells, primary rat mesenteric SMCs (pSMCs), and 6B5N cells, a clone of A7r5 cells expressing higher connexin43 (Cx43) to Cx40 ratio. Mechanical stimulation induced an intracellular Ca(2+) wave in pSMC and 6B5N cells that propagated to neighboring cells, whereas Ca(2+) waves in A7r5 cells failed to progress to neighboring cells. We demonstrate that Cx43 forms the functional GJs that are involved in mediating intercellular Ca(2+) waves and that co-expression of Cx40 with Cx43, depending on their expression ratio, may interfere with Cx43 GJ formation, thus altering junctional communication.

Artificial muscle: the human chimera is the future.

Severe heart failure and cerebral stroke are broadly associated with the impairment of muscular function that conventional treatments struggle to restore. New technologies enable the construction of "smart" materials that could be of great help in treating diseases where the main problem is muscle weakness. These materials "behave" similarly to biological systems, because the material directly converts energy, for example electrical energy into movement. The extension and contraction occur silently like in natural muscles. The real challenge is to transfer this amazing technology into devices that restore or replace the mechanical function of failing muscle. Cardiac assist devices based on artificial muscle technology could envelope a weak heart and temporarily improve its systolic function, or, if placed on top of the atrium, restore the atrial kick in chronic atrial fibrillation. Artificial sphincters could be used to treat urinary incontinence after prostatectomy or faecal incontinence associated with stomas. Artificial muscles can restore the ability of patients with facial paralysis due to stroke or nerve injury to blink. Smart materials could be used to construct an artificial oesophagus including peristaltic movement and lower oesophageal sphincter function to replace the diseased oesophagus thereby avoiding the need for laparotomy to mobilise stomach or intestine. In conclusion, in the near future, smart devices will integrate with the human body to fill functional gaps due to organ failure, and so create a human chimera.

Artificial muscle for end-stage heart failure.

We describe a device made of artificial muscle for the treatment of end-stage heart failure as an alternative to current heart assist devices. The key component is a matrix of nitinol wires and aramidic fibers called Biometal muscle (BM). When heated electrically, it produces a motorless, smooth, and lifelike motion. The BM is connected to a carbon fiber scaffold, tightening the heart and providing simultaneous assistance to the left and right ventricles. A pacemaker-like microprocessor drives the contraction of the BM. We tested the device in a dedicated bench model of diseased heart. It generated a systolic pressure of 75 mm Hg and ejected a maximum of 330 ml/min, with an ejection fraction of 12%. The device required a power supply of 6 V, 250 mA. This could be the beginning of an era in which BMs integrate or replace the mechanical function of natural muscles.

Assessment of quadriceps muscle inactivation with a new electrical stimulation paradigm.

INTRODUCTION: In this study we evaluated the validity of garment-based quadriceps stimulation (GQS) for assessment of muscle inactivation in comparison with femoral nerve stimulation (FNS). METHODS: Inactivation estimates (superimposed doublet torque), self-reported discomfort, and twitch and doublet contractile properties were compared between GQS and FNS in 15 healthy subjects. RESULTS: Superimposed doublet torque was significantly lower for GQS than for FNS at 20% and 40% maximum voluntary contraction (MVC) (P < 0.01), but not at 60%, 80%, and 100% MVC. Discomfort scores were systematically lower for GQS than for FNS (P < 0.05). Resting twitch and doublet peak torque were lower for GQS, and time to peak torque was shorter for GQS than for FNS (P < 0.01). CONCLUSIONS: GQS can be used with confidence for straightforward evaluation of quadriceps muscle inactivation, whereas its validity for assessment of contractile properties remains to be determined. Muscle Nerve 51: 117-124, 2015.

CARDIAC AND MUSCLE CHANNELOPATHIES: ROLES AND REGULATION OF VOLTAGE-GATED SODIUM CHANNELS

Abstract :The contraction of the heart or skeletal muscles is mainly due to the propagation, through excitable cells, of an electrical influx called action potential (AP). The AP results from the sequential opening of ion channels that generate inward or outward currents through the cell membrane. Among all the channels involved, the voltage-gated sodium channel is responsible for the rising phase of the action potential. Ten genes encode the different isoforms of these channels (from Nav1.1 to Nav1.9 and an atypical channel named NavX). Nav1.4 and Nav1.5 are the main skeletal muscle and cardiac sodium channels respectively. Their importance for muscle and heart function has been highlighted by the description of mutations in their encoding genes SCN4A and SCNSA. They lead respectively to neuromuscular disorders such as myotonia or paralysis (for Nav1.4), and to cardiac arrhythmias that can deteriorate into sudden cardiac death (for Nav1.5).The general aim of my PhD work has been to study diseases linked with channels dysfunction, also called channelopathies. In that purpose, I investigated the function and the regulation of the muscle and cardiac voltage-gated sodium channels. During the two first studies, I characterized the effects of two mutations affecting Nav1.4 and Nav1.5 function. I used the HEK293 model cells to express wild-type or mutant channels and then studied their biophysical properties with the patch-clamp technique, in whole cell configuration. We found that the SCN4A mutation produced complex alterations of the muscle sodium channel function, that could explain the myotonic phenotype described in patients carrying the mutation. In the second study, the index case was an heterozygous carrier of a SCNSA mutation that leads to a "loss of function" of the channel. The decreased sodium current measured with mutated Nay 1.5 channels, at physiological temperature, was a one of the factors that could explain the observed Brugada syndrome. The last project aimed at identifying a new potential protein interacting with the cardiac sodium channel. We found that the protein SAP97 binds the three last amino-acids of the C-terminus of Na,, 1.5. Our results also indicated that silencing the expression of SAP97 in HEK293 cells decreased the sodium current. Sodium channels lacking their three last residues also produced a reduced INa. These preliminary results suggest that SAP97 is implicated in the regulation of sodium channel. Whether this effect is direct or imply the action of an adaptor protein remains to be investigated. Moreover, our group has previously shown that Nav1.5 channels are localized to lateral membranes of cardiomyocytes by the dystrophin multiprotein complex (DMC). This suggests that sodium channels are distributed in, at least, two different pools: one targeted at lateral membranes by DMC and the other at intercalated discs by another protein such as SAP97.These studies reveal that cardiac and muscle diseases may result from ion channel mutations but also from regulatory proteins affecting their regulation.Résumé :La contraction des muscles et du coeur est principalement due à la propagation, à travers les cellules excitables, d'un stimulus électrique appelé potentiel d'action (PA). C'est l'ouverture séquentielle de plusieurs canaux ioniques transmembranaires, permettant l'entrée ou la sortie d'ions dans la cellule, qui est à l'origine de ce PA. Parmi tous les canaux ioniques impliqués dans ce processus, les canaux sodiques dépendant du voltage sont responsables de la première phase du potentiel d'action. Les différentes isoformes de ces canaux (de Nav1.1 à Nav1.9 et NavX) sont codées par dix gènes distincts. Nav1.4 et Nav1.5 sont les principaux variants exprimés respectivement dans le muscle et le coeur. Plusieurs mutations ont été décrites dans les gènes qui codent pour ces deux canaux: SCN4A (pour Nav1.4) et SCNSA (pour Nav1.5). Elles sont impliquées dans des pathologies neuromusculaires telles que des paralysies ou myotonies (SCN4A) ou des arythmies cardiaques pouvant conduire à la mort subite cardiaque (SCNSA).Mon travail de thèse a consisté à étudier les maladies liées aux dysfonctionnements de ces canaux, aussi appelées canalopathies. J'ai ainsi analysé la fonction et la régulation des canaux sodiques dépendant du voltage dans le muscle squelettique et le coeur. A travers les deux premières études, j'ai ainsi pu examiner les conséquences de deux mutations affectant respectivement les canaux Nav1.4 et Nav1.5. Les canaux sauvages ou mutants ont été exprimés dans des cellules HEK293 afin de caractériser leurs propriétés biophysiques par la technique du patch clamp en configuration cellule entière. Nous avons pu déterminer que la mutation trouvée dans le gène SCN4A engendrait des modifications importantes de la fonction du canal musculaire. Ces altérations fournissent des indications nous permettant d'expliquer certains aspects de la myotonie observée chez les membres de la famille étudiée. Le patient présenté dans la deuxième étude était hétérozygote pour la mutation identifiée dans le gène SCNSA. La perte de fonction des canaux Nav1.5 ainsi engendrée, a été observée lors d'analyses à températures physiologiques. Elle représente l'un des éléments pouvant potentiellement expliquer le syndrome de Brugada du patient. La dernière étude a consisté à identifier une nouvelle protéine impliquée dans la régulation du canal sodique cardiaque. Nos expériences ont démontré que les trois derniers acides aminés de la partie C-terminale de Nav1.5 pouvaient interagir avec la protéine SAP97. Lorsque que l'expression de la SAP97 est réduite dans les cellules HEK293, cela induit une baisse importante du courant sodique. De même, les canaux tronqués de leurs trois derniers acides aminés génèrent un flux ionique réduit. Ces résultats préliminaires suggèrent que SAP97 est peut-être impliquée dans la régulation du canal Na,,1.5. Des expériences complémentaires permettront de déterminer si ces deux protéines interagissent directement ou si une protéine adaptatrice est nécessaire. De plus, nous avons préalablement montré que les canaux Nav1.5 étaient localisés au niveau de la membrane latérale des cardiomyocytes par le complexe multiprotéique de la dystrophine (DMC). Ceci suggère que les canaux sodiques peuvent être distribués dans un minimum de deux pools, l'un ciblé aux membranes latérales pax le DMC et l'autre dirigé vers les disques intercalaires par des protéines telles que SAP97.L'ensemble de ces études met en évidence que certaines maladies musculaires et cardiaques peuvent être la conséquence directe de mutations de canaux ioniques, mais que l'action de protéines auxiliaires peut aussi affecter leur fonction.