NIH subventions R01 HL085369.

1. Préparation avancée des solutions de stock <ol><li> Préparer deux solutions de stock de solution de Tyrode (16x) à l&#39;avance dans l&#39;eau déminéralisée et de les stocker à 4 ° C: <ol class="lalpha"><li> Stock I (119.872 g / L de NaCl, 3,056 g / L de CaCl 2 (2H 2 O), 5,6 g / L de KCl, 2,6274 g / L NaH 2 PO 4, 3,408 g / L MgCl 2 (6H 2 O), (Fisher scientifique, Fair Lawn, NJ)); </li><li> Stock II (26,88 g / l NaHCO3, (Fisher Scientific, Fair Lawn, NJ)). </li></ol></li><li> Préparer des solutions stock de colorants fluorescents. Pour éviter la congélation et décongélation répétées, nous stockons 30 aliquotes des deux colorants à -20 ° C, ce qui est suffisant pour une expérience: <ol class="lalpha"><li> Sensibles au voltage solution stock de colorants RH237 (Invitrogen, Carlsbad, CA), 1,25 mg / ml solution dans le diméthylsulfoxyde (DMSO, Sigma, St. Louis, MO); </li><li> Indicateur de calcium Rhod-deux heures solution stock (Invitrogen, Carlsbad, CA), 1 mg / ml solution dans le DMSO. </li></ol></li><li> Préparer excitation-contraction solution stock découplant blebbistatin (Bioscience Tocris, St. Louis, MO, 2 mg / ml solution dans le DMSO) à l&#39;avance et de stocker les blebbistatin dissous à 4 ° C. </li></ol> 2. Préparer des solutions de perfusion et de l&#39;installation expérimentale 7 <ol start="4"><li> Fraîchement préparer la solution de Tyrode 2L de (128.2mM NaCl, CaCl 2 1,3 mm (2H 2 O), 4,7 KCl, MgCl 2 1,05 mm (6H 2 O), 1.19mM NaH 2 PO 4, 20mm de NaHCO 3, 11.1mm D-glucose dans l&#39;eau, pH = 7,35 ± désionisée 0,05). Si la solution stock est utilisé pour faire des 2L solution de Tyrode (suffisant pour une expérience) de prendre 1750 ml d&#39;eau déminéralisée et mélanger dans 125 ml de bouillon de I, 125 ml de bouillon de II, et 4g de glucose. </li><li> Allumez les deux pompes du système de perfusion. Régler la pompe péristaltique (Peri-Star, WPI, Sarasota, Etats-Unis) qui est utilisé pour perfusion rétrograde à 40 mL / min. Réglez la pompe péristaltique d&#39;autres (Cole-Parmer Masterflex Pompe Peristalic L / S, Instrument Company Cole-Parmer, Vernon Hills, Illinois) qui est utilisé pour surfusion et de revenir le perfusat de retour au réservoir tenant à 80 mL / min. </li><li> Lavez le système de perfusion à l&#39;éthanol 70% pendant 30 min puis à l&#39;eau déminéralisée 2L. </li><li> Une fois toute l&#39;eau déminéralisée est évacué de la chambre, faire circuler la solution de Tyrode et le passer à travers un filtre de 5 um (Millipore, Billerica, MA, Etats-Unis). Chauffer le perfusat à 37 ° C avec une chemise d&#39;eau et circulateur (ThermoNESLAB EX7, Newtown, Etats-Unis) et d&#39;oxygéner le perfusat par barbotage d&#39;O 2 / CO 2 (95% / 5%) de gaz dans la solution. Surveiller le pH de la solution avec un pH-mètre (Oakton Instruments, Vernon Hills, IL) et d&#39;ajuster le taux d&#39;O 2 / CO 2 bouillonnant à maintenir le pH à 7,35 ± 0,05. Poursuivre la surveillance du pH et de température pendant l&#39;expérience. </li><li> L&#39;appareil de cartographie optique double composé de deux caméras MICAM Ultima-L CMOS (SciMedia, Costa Mesa, Californie) qui ont spatiale élevée (100x100 pixels, 230 ± 20 um par pixel) et temporelle (1000-3000 images / sec) de résolution. Correction d&#39;un filtre passe-bande (590 ± 15 nm, Thorlabs, Newton, NJ), en face de la caméra d&#39;imagerie calcique désigné, tandis que, d&#39;un filtre passe-temps (&gt; 700 nm, Thorlabs, Newton, NJ), doit être placé dans devant la caméra d&#39;imagerie de tension désigné. Les caméras sont disposées perpendiculairement les uns aux autres par un porteur, qui contient un miroir dichroïque (635 nm de coupure, Omega Optical, Brattleboro, VT). Immédiatement au-dessous du support de caméra à double il ya un objectif (Nikon Nikkor 55 mm 1:1.4 235 052), qui focalise la lumière d&#39;émission provenant du coeur sur le miroir dichroïque. Le travail à distance est d&#39;environ 3cm. La lumière d&#39;excitation est produite par une lampe halogène (Newport Oriel Instruments, Stratford, CT; SciMedia, Costa Mesa, Californie) et est passé à travers un filtre de chaleur, d&#39;obturation et filtre passe-bande (520 ± 45 nm). Un guide de lumière flexibles dirige la lumière filtrée passe-bande sur la préparation, et d&#39;un obturateur est utilisée pour s&#39;assurer que la préparation est exposé à la lumière que lors de l&#39;acquisition d&#39;image pour éviter photoblanchiment des colorants. </li><li> Préparer Ag / AgCl 2 électrodes de stimulation et de détection à l&#39;avance et de les installer dans la chambre avant de placer au cœur. Assurez-vous que les amplificateurs et les filtres sont ajustés à des niveaux appropriés. </li></ol> 3. Récolte au cœur de la souris, cathétériser, et mettre en place une perfusion de Langendorff <ol start="10"><li> Anesthésier la souris avec la kétamine / xylazine (kétamine, 80mg/kg de poids corporel; xylazine, 10 mg / kg de poids corporel) et l&#39;héparine (100 unités) par injection intrapéritonéale. Assurer le niveau approprié de l&#39;anesthésie par le manque de réflexe de douleur. </li><li> Après une incision médiane du sternum, enlever rapidement le cœur et le laver dans oxygénée (95% d&#39;O 2, 5% CO 2), à température constante (37 ± 1 ° C) une solution de Tyrode. </li><li> En utilisant un microscope à dissection, D&#39;identifier rapidement l&#39;aorte et de faire une coupe nette dans l&#39;aorte ascendante dessous de l&#39;artère sous-clavière droite. Une courte section de l&#39;aorte est ensuite attaché à une coutume fait de calibre 21 canule avec un embout évasé. 4-0 suture de soie noire tressée (Surgical Corporation Spécialités, Reading, PA) est utilisé pour fixer le cœur sur la canule. Après canulation, le coeur est perfusé et rétrograde perfusées avec une solution de Tyrode. Le taux de perfusion rétrograde est ajusté dans la gamme de 2-5 mL / min pour maintenir la pression aortique entre 60 et 80 mmHg (capteur de pression, instruments de précision du Monde Inc (WPI), Sarasota, Etats-Unis; Pont Amplificateur TBM4M, WPI, Sarasota, Etats-Unis). </li><li> Après le cœur est une canule, du poumon, du thymus et des tissus adipeux sont ensuite disséqués et enlevé. </li><li> Le cœur isolé est épinglé (Outils Fine Science) à l&#39;apex au fond de la chambre de perfusion (couché Sylgard) pour empêcher flux induits par le mouvement. Les appendices auriculaires droite et gauche sont également étirées et épinglé (Outils Fine Science, Inc, Foster City, CA) au fond de la chambre, qui donne la superficie maximale pour les mesures optiques de l&#39;oreillette. Très important! Un tube de silicium petite est inséré dans le ventricule gauche par les veines pulmonaires et fixé par une suture en soie au tissu conjonctif voisin. Cela évite la congestion solution et l&#39;acidification de l&#39;perfusat piégés dans le ventricule gauche, qui est particulièrement important après la suppression des contractions ventriculaires avec un découplant excitation-contraction (voir partie 4 étape 19). </li><li> Une électrode sur mesure est placé sur la surface du cœur de mener les stimulations rythme, qui est généré par le maître-8 (AMP Instruments Ltd, Jérusalem, Israël) ou PowerLab 26T (AD Instruments, Sydney, Australie). </li><li> Un couvercle en verre petite est fixé sur la surface de la solution sur le cœur pour réduire artefacts de mouvement de la solution de vibration. </li><li> Focaliser la lumière d&#39;excitation sur le cœur. En outre, ajuster la distance entre l&#39;appareil photo et le double cœur pour que la résolution maximale sera obtenue. </li><li> Eteignez toutes les lumières dans la pièce et de commencer les enregistrements électriques utilisant PowerLab 26T. </li></ol> 4. Tension de charge et de colorants sensible au calcium et d&#39;excitation-contraction découplant <ol start="19"><li> Warm-up 0,6 mL d&#39;blebbistatin. Mélanger 0,5 mL de la blebbistatin avec le perfusat dans le réservoir de maintien. Diluer le reste 0,1 mL de blebbistatin dans 1 ml de solution de Tyrode et de l&#39;injecter lentement (sur une période de 20 minutes) par un port de drogue situé près de la canule. Observez que blebbistatin réduit progressivement l&#39;artefact de mouvement. </li><li> Diluer 30 uL de la tension de la solution de teinture d&#39;actions sensibles RH237 dans la solution 1 ml de Tyrode et injecter lentement au cours de 5-7 minutes dans le port d&#39;injection même blebbistatin. </li><li> 30 uL de calcium indicateur de Rhod-deux heures est de 1:1 mélangé avec Pluronic F127 (Invitrogen, Carlsbad, CA, solution à 20% dans le DMSO), puis dilué dans 1 mL de solution de Tyrode et lentement appliqué sur 5-10 min à travers le port même injection . </li><li> Attendre 5-10 minutes pour blebbistatin et colorants pour atteindre la membrane cellulaire et le cytosol. Continuer avec le protocole lorsque le mouvement est complètement supprimée. </li><li> Surveiller en permanence les enregistrements ECG au cours de la procédure entière pour assurer le fonctionnement normal électrique du cœur. </li><li> Commencer enregistrements signal fluorescent en utilisant les logiciels personnalisés SciMedia (SciMedia, Costa Mesa, Californie). </li></ol> 5. Les résultats représentatifs: <img alt="Figure 1" src="/files/ftp_upload/3275/3275fig1.jpg" /> Figure 1. Dispositif expérimental pour la perfusion, les enregistrements électriques et optiques de cartographie. EM = émission; Lp = longpass <img alt="Figure 2" src="/files/ftp_upload/3275/3275fig2.jpg" /> Figure 2. Préparation expérimentale et des exemples de signaux enregistrés pendant la stimulation ventriculaire. Gauche: Les signaux ECG sont recueillies auprès de Ag / AgCl 2 électrodes à disque (en haut) et un exemple de S1S1 protocole de stimulation est indiqué (en bas). Centre: préparation de Langendorff cœur de la souris. Droite: Représentant des potentiels d&#39;action optiques et signaux calciques transitoires d&#39;oreillettes (en haut) et les ventricules (bas) sont affichés. La flèche jaune (en haut) les points de la dispersion du signal fluorescent provenant des ventricules, ce qui est vu dans les enregistrements auriculaire. LV = ventricule gauche; VD = ventricule droit; LA = oreillette gauche; RA = oreillette droite <img alt="Figure 3" src="/files/ftp_upload/3275/3275fig3.jpg" /> Figure 3. Représentant enregistrements optiques de V m et CAT des ventricules du cœur de souris de type sauvage. A. La préparation expérimentale avec un tableau de points uniformément répartis marquée par des points noirs dont les enregistrements optiques peut être vu dans (C). B. Exemple de traçage de V m et chat d&#39;un emplacement central sur le tableau (voir encadré In (C)). C. V m (bleu) et Cat (rouge) à partir du tableau des emplacements régulièrement espacés. Les signaux ont été mis en cellule de 3x3. <img alt="Figure 4" src="/files/ftp_upload/3275/3275fig4.jpg" /> Figure 4. Carte d&#39;activation et de conduction. A. Une carte d&#39;activation par exemple d&#39;un cœur de souris de type sauvage avec transversale (T) et longitudinale (L) directions indiquées par les flèches blanches. B. V signaux m (haut) et dV / dt (Bas), correspondant aux trois points de vue dans un (T1, T2, T3, L1, L2, L3). <img alt="Figure 5" src="/files/ftp_upload/3275/3275fig5.jpg" /> Figure 5. Potentiels d&#39;action et analyse de la durée de calcium transitoire. A. Durée du potentiel d&#39;action à 80% de repolarisation (APD80) et la durée de calcium transitoire à 80% détente (80 CAD) les cartes sont affichées à partir d&#39;un coeur dans des conditions de contrôle (à gauche) et après application 30 nM isoprotérénol (à droite). La couleur jaune / vert dans les ventricules (à droite) indique l&#39;isoprotérénol raccourcie APD80 et 80 CAD. B. Exemple de tracés et de 80 CAD APD80 des ventricules souris de type sauvage (haut) et oreillettes (bas).

<ol>
	<li>Efimov, I. R., Rendt, J. M., Salama, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+maps+of+intracellular+[Ca2+]i+transients+and+action-potentials+from+the+surface+of+perfused+guinea-pig+hearts.">Optical maps of intracellular [Ca2+]i transients and action-potentials from the surface of perfused guinea-pig hearts.</a> Circulation. 90, 1-1 (1994).</li><li>Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+mapping+of+repolarization+and+refractoriness+from+intact+hearts.">Optical mapping of repolarization and refractoriness from intact hearts.</a> Circulation. 90, 1469-1480 (1994).</li><li>Choi, B. R., Salama, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+maps+of+optical+action+potentials+and+calcium+transients+in+guinea-pig+hearts:+mechanisms+underlying+concordant+alternans.">Simultaneous maps of optical action potentials and calcium transients in guinea-pig hearts: mechanisms underlying concordant alternans.</a> J Physiol. 529, 171-188 (2000).</li><li>Pruvot, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+calcium+cycling+versus+restitution+in+the+mechanism+of+repolarization+alternans.">Role of calcium cycling versus restitution in the mechanism of repolarization alternans.</a> Circ Res. 94, 1083-1090 (2004).</li><li>Efimov, I. R., Nikolski, V. P., Salama, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+the+heart.">Optical imaging of the heart.</a> Circ Res. 95, 21-33 (2004).</li><li>Fast, V. G., Dhein, S., Delmoar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recording+action+potential+using+voltage+sensitive+dyes.">Recording action potential using voltage sensitive dyes.</a> Practical methods in cardiovascular research. , 233-255 (2005).</li><li>Glukhov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+K(ATP)+channel+pharmacology+in+intact+mouse+heart.">Differential K(ATP) channel pharmacology in intact mouse heart.</a> J Mol Cell Cardiol. , (2009).</li><li>Baker, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+dispersion+of+repolarization+and+refractoriness+in+transgenic+mouse+hearts+promotes+reentrant+ventricular+tachycardia.">Enhanced dispersion of repolarization and refractoriness in transgenic mouse hearts promotes reentrant ventricular tachycardia.</a> Circ Res. 86, 396-407 (2000).</li><li>Sutherland, F. J., Hearse, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolated+blood+and+perfusion+fluid+perfused+heart.">The isolated blood and perfusion fluid perfused heart.</a> Pharmacol Res. 41, 613-627 (2000).</li><li>Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virtual+electrode+polarization+in+the+far+field:+implications+for+external+defibrillation.">Virtual electrode polarization in the far field: implications for external defibrillation.</a> Am J Physiol Heart Circ Physiol. 279, 1055-1070 (2000).</li><li>Hammouda, M., Kinosita, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+coronary+circulation+in+the+isolated+heart.">The coronary circulation in the isolated heart.</a> J Physiol. 61, 615-628 (1926).</li><li>Salama, G., Hwang, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+optical+mapping+of+intracellular+free+calcium+and+action+potentials+from+Langendorff+perfused+hearts.">Simultaneous optical mapping of intracellular free calcium and action potentials from Langendorff perfused hearts.</a> Curr Protoc Cytom. 12, 17-17 (2009).</li><li>Lou, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+panoramic+imaging+of+epicardial+electrical+activity.">Quantitative panoramic imaging of epicardial electrical activity.</a> Ann Biomed Eng. 36, 1649-1658 (2008).</li><li>Salama, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+new,+long-wavelength,+voltage-sensitive+dyes+in+the+heart.">Properties of new, long-wavelength, voltage-sensitive dyes in the heart.</a> J Membr Biol. 208, 125-140 (2005).</li></ol>

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Dans cette expérience, nous avons modifié la méthode de perfusion de Langendorff en ajoutant un tube de silicium faible, ce qui est particulièrement crucial après la suppression des contractions ventriculaires avec un découplant excitation-contraction. Le tube de silicium est utilisé pour prévenir la congestion solution, l&#39;acidification de la solution de perfusion, et le développement de l&#39;ischémie dans le ventricule gauche. Le cœur de la souris est très sensible à l&#39;hypothermie; ainsi, les variations de température à travers le cœur d&#39;entraîner des variations artificielles dans la durée du potentiel d&#39;action. Par conséquent, un système de chauffage a été mis en œuvre dans la chambre de perfusion afin de maintenir une température constante de 37 ° C pendant la totalité de l&#39;expérience 8. Depuis un modèle de Langendorff ne conserve pas l&#39;innervation du cœur, on doit envisager d&#39;ajouter neurotransmetteurs pour le perfusat afin d&#39;enquêter physiologiques du tonus sympathique et parasympathique 9. Outre une perfusion rétrograde, plus de surfusion du cœur aide à maintenir adaptée paramètres environnementaux tels que le pH et la température. Dans cette méthode, le coeur perfusé de Langendorff a été placée horizontalement. Une configuration de Langendorff verticales perfusion peut également être utilisé 10, mais peut entraîner un peu différent mécanique cardiaque 11. En plus de caméras CMOS, des détecteurs de rechange sont également disponibles et peuvent être appliquées à la carte V m et chat simultanément 12. Application de caméras CMOS de haute résolution spatio-temporelle assure la précision des enregistrements, mais les signaux optiques ne sont pas la cartographie d&#39;une seule cellule. Au contraire, chaque signal de fluorescence provient de centaines ou de milliers de cellules, en fonction de grossissement optique. La fluorescence du ventricule beaucoup plus grand peut fausser les signaux auriculaire par diffusion optique, par conséquent, une interprétation prudente des signaux enregistrés optiquement est nécessaire. Une autre limitation de la préparation de la souris est la distorsion du signal et le bruit induit par la courbure de la surface en raison de la petite taille du cœur 13. Mesures de vitesse de conduction peut être modifié non seulement par la courbure de la souris, mais aussi le cœur de la polarité des électrodes et des électrodes virtuel. Pour atteindre la précision pour la vitesse de conduction, l&#39;anisotropie d&#39;activation, et des cartes de repolarisation, de rectification et de focalisation de la caméra à la surface du cœur est essentielle. Dans cette méthode, en temps réel les enregistrements ECG peuvent compléter la recherche optique de l&#39;électrophysiologie cardiaque. Tension colorant sensible (RH237) et l&#39;indicateur de calcium (Rhod-deux heures) sont utilisés dans le protocole en raison de leur réponse rapide, d&#39;excitation semblables, et les spectres d&#39;émission distincts 3,7. Il ya d&#39;autres combinaisons de colorants qui peuvent être utilisés pour mesurer V m et CAT autres que RH237 et Rhod-2 heures 3. Un roman sensible au voltage colorant, PGHI, avec un grand déplacement de Stoke (&gt; 200nm) a été trouvée pour permettre une meilleure m V et les signaux de chat car la plus grande séparation des longueurs d&#39;onde d&#39;émission entre PGHI et Rhod-2 heures 14. Les améliorations futures peuvent se concentrer sur l&#39;exploration de nouvelles sondes fluorescentes, le développement de détecteurs d&#39;imagerie, et l&#39;amélioration des logiciels de traitement d&#39;image. Une résolution et une nouvelle imagerie optique pour la cartographie 3D optique sont aussi importants les orientations futures de la cartographie optique 5.

<table border="1"><tbody><tr><td> Chimique </td><td> Société </td><td> Nombre de catalogue </td></tr><tr><td> NaCl </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> S271-1 </td></tr><tr><td> CaCl 2 (2H 2 O) </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> C79-500 </td></tr><tr><td> KCl </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> S217-500 </td></tr><tr><td> MgCl 2 (6H 2 O) </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> M33-500 </td></tr><tr><td> NaH 2 PO 4 (H 2 O) </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> S369-500 </td></tr><tr><td> NaHCO 3 </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> S233-3 </td></tr><tr><td> D-glucose </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> D16-1 </td></tr><tr><td> Blebbistatin </td><td> Bioscience Tocris, Ellisville, MO </td><td> 1760 </td></tr><tr><td> RH237 </td><td> Invitrogen, Carlsbad, CA </td><td> S1109 </td></tr><tr><td> Rhod-deux heures </td><td> Invitrogen, Carlsbad, CA </td><td> R1244 </td></tr><tr><td> Pluronic F127 </td><td> Invitrogen, Carlsbad, CA </td><td> P3000MP </td></tr><tr><td> Diméthylsulfoxyde (DMSO) </td><td> Sigma, St. Louis, MO </td><td> D2650 </td></tr></tbody></table>

optical mapping of action potentials and calcium transients in the mouse heart

Le cœur de la souris est un modèle populaire pour les études cardiovasculaires en raison de l&#39;existence d&#39;une technologie à faible coût pour le génie génétique dans cette espèce. Phénotypage cardiovasculaire physiologique du cœur de souris peut être facilement fait en utilisant l&#39;imagerie de fluorescence employant différentes sondes de potentiel transmembranaire (V m), les transitoires de calcium (CAT), et d&#39;autres paramètres. Couplage excitation-contraction est caractérisée par une dynamique d&#39;action du calcium intracellulaire potentiel et, par conséquent, il est extrêmement important de cartographier la fois V m et chat simultanément au même endroit sur ​​le cœur 1-4. Simultanée de cartographie optique de Langendorff perfusé coeurs de souris a le potentiel pour élucider les mécanismes qui sous-tendent l&#39;insuffisance cardiaque, les arythmies, les maladies métaboliques, et d&#39;autres maladies cardiaques. Visualisation de l&#39;activation, la vitesse de conduction, la durée du potentiel d&#39;action, et d&#39;autres paramètres à une myriade de sites ne peuvent pas être atteints de l&#39;enquête au niveau cellulaire, mais est bien résolu par cartographie optique 1,5,6. Dans cet article nous présentons la configuration d&#39;instrumentation et des conditions expérimentales pour la cartographie optique simultanée de V m et chat dans les cœurs de souris à haute résolution spatio-temporelle en utilisant l&#39;état de l&#39;art technologie d&#39;imagerie CMOS. Conformément enregistrements optiques obtenus avec cette méthode montrent que la cartographie simultanée optique de coeurs perfusés de Langendorff la souris est à la fois réalisable et fiable.

Watch this Scientific Journal Video about Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart at JoVE.com

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

Ce document détaille la procédure de dissection, la configuration instrumentale, et des conditions expérimentales lors de la cartographie optique de potentiel transmembranaire (Vm) et de calcium intracellulaire transitoire (CAT) dans Langendorff isolés perfusés intacte cœur de la souris.

Cartographie optique de potentiels d&#39;action et les transitoires de calcium dans le cœur de la souris

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

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27.0K vues.  Washington University in St. Louis. Ce document détaille la procédure de dissection, la configuration instrumentale, et des conditions expérimentales lors de la cartographie optique de potentiel transmembranaire (Vm) et de calcium intracellulaire transitoire (CAT) dans Langendorff isolés perfusés intacte cœur de la souris.

Vidéo: Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Tridimensionnelle optique résolution microscopie photoacoustique

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

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Bio-ingénierie

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Multiparamétrique cartographie optique du Cœur lapin perfusé de Langendorff

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

Une approche recombinaison Cre-Lox P pour la détection de la fusion cellulaire In Vivo</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

En synthèse in vitro de la maternelle, de l&#39;espacement de longue fibreux et segmentaire collagène Espacement long

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

Détection optique de<em&gt; E. coli</em&gt; Bactérie par mésoporeux Silicon biocapteurs

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

En cartographie in situ des propriétés mécaniques des biofilms par Microrhéologie de particules de suivi

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Préparation de la 3D collagène Gels et microcanaux pour l&#39;étude des interactions 3D<em&gt; In Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

Microscopie de Fluorescence lumière-feuille pour capturer des Images 4-dimensionnelle des effets de modulation de la contrainte de cisaillement sur le coeur de poisson-zèbre en développement

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. Traditional light-sheet techniques involve the use of a single illumination beam directed orthogonally at sample tissue. Images of large samples that are produced using a single illumination beam contain stripes or artifacts and suffer from a reduced resolution due to the scattering and absorption of light by the tissue. This study uses a dual-sided illumination beam and a simplified CLARITY optical clearing technique for the murine heart. These techniques allow for deeper imaging by removing lipids from the heart and produce a large field of imaging, greater than 10 x 10 x 10 mm3. As a result, this strategy enables us to quantify the ventricular dimensions, track the cardiac lineage, and localize the spatial distribution of cardiac-specific proteins and ion-channels from the post-natal to adult mouse hearts with sufficient contrast and resolution.

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

Microscopie de Fluorescence lumière-feuille pour l’étude du coeur murin

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. ...

Biaxial Mechanical Characterizations of Atrioventricular Heart Valves

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive models, which provide a mathematical representation of the mechanical function of those structures. This presented biaxial mechanical testing protocol involves (i) tissue acquisition, (ii) the preparation of tissue specimens, (iii) biaxial mechanical testing, and (iv) postprocessing of the acquired data. First, tissue acquisition requires obtaining porcine or ovine hearts from a local Food and Drug Administration-approved abattoir for later dissection to retrieve the valve leaflets. Second, tissue preparation requires using tissue specimen cutters on the leaflet tissue to extract a clear zone for testing. Third, biaxial mechanical testing of the leaflet specimen requires the use of a commercial biaxial mechanical tester, which consists of force-controlled, displacement-controlled, and stress-relaxation testing protocols to characterize the leaflet tissue&#39;s mechanical properties. Finally, post-processing requires the use of data image correlation techniques and force and displacement readings to summarize the tissue&#39;s mechanical behaviors in response to external loading. In general, results from biaxial testing demonstrate that the leaflet tissues yield a nonlinear, anisotropic mechanical response. The presented biaxial testing procedure is advantageous to other methods since the method presented here allows for a more comprehensive characterization of the valve leaflet tissue under one unified testing scheme, as opposed to separate testing protocols on different tissue specimens. The proposed testing method has its limitations in that shear stress is potentially present in the tissue sample. However, any potential shear is presumed negligible.

This protocol involves characterizations of atrioventricular valve leaflets with force-controlled, displacement-controlled, and stress-relaxation biaxial mechanical testing procedures. Results acquired with this protocol can be used for constitutive model development to simulate the mechanical behavior of functioning valves under a finite element simulation framework.

Biaxiales caractérisations mécaniques des valvules auriculo-ventriculaire

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive ...