NIH concede R01 HL085369.

1. Preparazione avanzata delle soluzioni madre <ol><li> Preparare due soluzioni stock di soluzione di Tyrode (16x) in anticipo in acqua deionizzata e conservarli a 4 ° C: <ol class="lalpha"><li> Magazzino I (119,872 g / L di NaCl, 3,056 g / L CaCl 2 (2H 2 O), 5,6 g / L di KCl, 2,6274 g / L NaH 2 PO 4, 3,408 g / L MgCl 2 (6H 2 O), (Fisher scientifico, Fair Lawn, New Jersey)); </li><li> Magazzino II (26,88 g / L NaHCO3, (Fisher Scientific, Fair Lawn, New Jersey)). </li></ol></li><li> Preparare soluzioni di coloranti fluorescenti. Per evitare congelamenti e scongelamenti ripetuti, memorizziamo 30 aliquote da ml di entrambi i coloranti a -20 ° C, sufficiente per un esperimento: <ol class="lalpha"><li> Voltaggio-sensibile colorante RH237 (Invitrogen, Carlsbad, CA) soluzione madre, 1,25 mg / ml soluzione in dimetilsolfossido (DMSO, Sigma, St. Louis, MO); </li><li> Calcio indicatore Rhod-02:00 (Invitrogen, Carlsbad, CA) soluzione madre, 1 mg / mL di soluzione in DMSO. </li></ol></li><li> Preparare eccitazione-contrazione blebbistatin soluzione disaccoppiatore magazzino (Bioscience Tocris, St. Louis, MO, 2 mg / mL di soluzione in DMSO) in anticipo e conservare il blebbistatin sciolto a 4 ° C. </li></ol> 2. Preparare le soluzioni di perfusione e sperimentale 7 <ol start="4"><li> Appena preparare la soluzione di Tyrode 2L (128.2mM NaCl, 1.3mm CaCl 2 (2H 2 O), 4,7 mm KCl, 1,05 mm MgCl 2 (6H 2 O), 1.19mM NaH 2 PO 4, 20 mM NaHCO 3, 11.1mM D-glucosio in acqua deionizzata, pH = 7,35 ± 0,05). Se la soluzione viene attualmente usato per fare 2L di soluzione di Tyrode (sufficiente per un esperimento) prendere 1750 ml di acqua deionizzata e mescolare in 125 ml di Stock I, 125 ml di Stock II e 4g di glucosio. </li><li> Accendere i due pompe dei sistemi di perfusione. Impostare la pompa peristaltica (Peri-Star, WPI, Sarasota, Stati Uniti) che viene utilizzato per la perfusione retrograda a 40 ml / min. Impostare l&#39;altra pompa peristaltica (Cole-Parmer Masterflex Pompa Peristalic L / S, Cole-Parmer Instrument Company, Vernon Hills, Illinois) che viene utilizzato per superfusione e di restituire il perfusato al serbatoio tenendo a 80 ml / min. </li><li> Lavare il sistema di perfusione con etanolo al 70% per 30 min e poi con 2 litri di acqua deionizzata. </li><li> Una volta che tutta l&#39;acqua deionizzata è evacuata dalla camera, far circolare la soluzione del Tyrode e passare attraverso un filtro a 5 micron (Millipore, Billerica, MA, USA). Scaldare il perfusato a +37 ° C con una camicia d&#39;acqua e circolatore (ThermoNESLAB EX7, Newtown, USA) e ossigenare il perfusato facendo gorgogliare O 2 / CO 2 (95% / 5%) del gas nella soluzione. Monitorare il pH della soluzione con un pH-metro (Oakton Instruments, Vernon Hills, IL) e regolare la velocità di O 2 / CO 2 bolle di mantenere il pH a 7,35 ± 0,05. Continua il monitoraggio del pH e temperatura durante l&#39;esperimento. </li><li> L&#39;apparecchio a doppia mappatura ottico è costituito da due MICAM Ultima-L telecamere CMOS (SciMedia, Costa Mesa, CA) che hanno un alto spaziale (100x100 pixel, 230 ± 20 micron per pixel) e temporale (1.000-3.000 fotogrammi / sec) risoluzione. Fissare un filtro passa-banda (590 ± 15 nm, Thorlabs, Newton, New Jersey) davanti alla telecamera calcio designato immagini, mentre, un filtro passa-lungo (&gt; 700 nm, Thorlabs, Newton, New Jersey) deve essere posizionato in davanti alla telecamera tensione designato imaging. Le telecamere sono disposti perpendicolarmente l&#39;uno all&#39;altro da titolare, che contiene uno specchio dicroico (635 nm di taglio, Omega Optical, Brattleboro, VT). Immediatamente sotto il supporto dual camera c&#39;è un obiettivo (Nikon NIKKOR 55 millimetri 1:1,4 235052), che focalizza la luce di emissione che viene dal cuore sullo specchio dicroico. La distanza di lavoro è di circa 3cm. La luce di eccitazione è generata da una lampada alogena (Newport Oriel Instruments, Stratford, CT; SciMedia, Costa Mesa, CA) ed è passato attraverso un filtro di calore, otturatore e filtro passa-banda (520 ± 45 nm). La guida di luce flessibile dirige il passa-banda luce filtrata sulla preparazione, e un otturatore è usato per garantire che la preparazione è esposto alla luce solo durante l&#39;acquisizione di immagini al fine di evitare photobleaching dei coloranti. </li><li> Preparare Ag / AgCl 2 elettrodi per la stimolazione e sensing in anticipo e installarli nella camera prima di mettere il cuore. Assicurarsi che gli amplificatori e filtri vengono regolate a livelli adeguati. </li></ol> 3. Raccolto il cuore del mouse, cannulate, e impostare la perfusione Langendorff <ol start="10"><li> Anestetizzare il mouse con ketamina / xylazina (ketamin, 80mg/kg di peso corporeo; xylazina, 10 mg / kg di peso corporeo) ed eparina (100 unità) mediante iniezione intraperitoneale. Assicurare un adeguato livello di anestesia dalla mancanza di riflesso il dolore. </li><li> Dopo una metà sternale incisione, rimuovere velocemente il cuore e lavarlo in ossigenata (95% O 2, 5% CO 2), a temperatura costante (37 ± 1 ° C) Tyrode la soluzione. </li><li> Utilizzando un microscopio da dissezione, Identificare rapidamente l&#39;aorta e fare un taglio netto tra l&#39;aorta ascendente sotto l&#39;arteria succlavia destra. Un breve tratto di aorta viene poi attaccato ad una misura da 21 gauge cannula con una punta svasata. 4-0 nero-sutura di seta intrecciata (Surgical Corporation Specialità, Reading, PA) viene utilizzata per fissare il cuore sulla cannula. Dopo incannulazione, il cuore è retrogrado e superfused perfuso con la soluzione di Tyrode. Il tasso di perfusione retrograda viene regolato nel range di 2-5 mL / min per mantenere la pressione aortica tra i 60 e 80 mmHg (trasduttore di pressione, World strumenti di precisione Inc (WPI), Sarasota, Stati Uniti d&#39;America; Ponte Amplificatore TBM4M, WPI, Sarasota, USA). </li><li> Dopo che il cuore è cannulata, polmone, timo, e tessuto adiposo vengono poi sezionati e rimossi. </li><li> Il cuore isolato è appuntato (Strumenti di scienze arti) al vertice al fondo della camera di perfusione (patinata Sylgard) per evitare flusso indotta movimento. Le appendici atriale destra e sinistra sono anche allungati e appuntato (Strumenti Scienza Fine, Inc, Foster City, CA) al fondo della camera, che offre massima area di superficie per misurazioni ottiche degli atri. Molto importante! Un tubo di silicone viene inserita nel ventricolo sinistro attraverso le vene polmonari e fissato dalla sutura di seta al vicino tessuto connettivo. Questa soluzione evita la congestione e l&#39;acidificazione del perfusato intrappolati nel ventricolo sinistro, che è particolarmente importante dopo la soppressione delle contrazioni del ventricolo con una eccitazione-contrazione disaccoppiatore (vedi parte 4 punto 19). </li><li> Un elettrodo di misura è posto sulla superficie del cuore di condurre la stimolazione stimoli, che viene generato dal Maestro-8 (AMP Instruments Ltd, Gerusalemme, Israele) o PowerLab 26T (AD Instruments, Sydney, Australia). </li><li> Un bicchiere piccolo coperchio è fissato sulla superficie della soluzione sul cuore per ridurre artefatti da movimento dalla soluzione vibrante. </li><li> Focalizzare la luce di eccitazione sul cuore. Inoltre, regolare la distanza tra l&#39;apparato doppia fotocamera e il cuore in modo che la risoluzione massima si ottiene. </li><li> Spegnere tutte le luci in sala e cominciare le registrazioni elettrica usando PowerLab 26T. </li></ol> 4. Tensione a vuoto e tinture di calcio sensibili e eccitazione-contrazione disaccoppiatore <ol start="19"><li> Warm-up 0.6 ml di blebbistatin. Mescolare 0,5 ml di blebbistatin con il perfusato nel serbatoio azienda. Diluire il restante 0,1 mL di blebbistatin in 1 ml di soluzione Tyrode e iniettare lentamente (in un periodo di 20 minuti) attraverso una porta di droga vicino alla cannula. Osservare come blebbistatin riduce gradualmente la artefatti da movimento. </li><li> Diluire 30 ml di tensione sensibili colorante RH237 soluzione madre in soluzione 1 ml Tyrode e iniettare lentamente oltre 5-7 minuti nella porta di iniezione stesso blebbistatin. </li><li> 30 microlitri di calcio indicatore Rhod-2 1:01 AM è mescolato con Pluronic F127 (Invitrogen, Carlsbad, CA, 20% soluzione in DMSO) e poi diluito in 1 mL di soluzione Tyrode e lentamente applicato su 5-10 min attraverso la porta stessa iniezione . </li><li> Attendere 5-10 minuti per blebbistatin e tinture per raggiungere la membrana cellulare e citosol. Continuare con il protocollo quando il movimento è completamente soppresso. </li><li> Monitorare costantemente le registrazioni ECG sopra l&#39;intera procedura per garantire la normale funzione elettrica del cuore. </li><li> Iniziare le registrazioni del segnale fluorescente SciMedia utilizzando il software personalizzato (SciMedia, Costa Mesa, CA). </li></ol> 5. Rappresentante dei risultati: <img alt="Figura 1" src="/files/ftp_upload/3275/3275fig1.jpg" /> Figura 1. Impostazione sperimentale per la perfusione, registrazioni elettrici e la mappatura ottica. EM = emissioni; Lp = longpass <img alt="Figura 2" src="/files/ftp_upload/3275/3275fig2.jpg" /> Figura 2. Preparazione sperimentale ed esempi segnale registrato durante la stimolazione ventricolare. A sinistra: segnali ECG sono raccolti direttamente presso Ag / AgCl 2 elettrodi disco (in alto) e un esempio di S1S1 protocollo di stimolazione è mostrato (in basso). Centro: preparazione del mouse Langendorff cuore. A destra: Rappresentante potenziali d&#39;azione ottica e segnali di calcio transitori da atri (Top) e ventricoli (basso) sono mostrati. La freccia gialla (Top) punti alla dispersione segnale fluorescente proveniente dal ventricoli, che si vede nelle registrazioni atriale. LV = ventricolo sinistro; RV = ventricolo destro; LA = atrio sinistro; RA = atrio destro <img alt="Figura 3" src="/files/ftp_upload/3275/3275fig3.jpg" /> Figura 3. Rappresentante registrazioni ottiche di V m e Cat dai ventricoli di un cuore selvaggio del mouse tipo. A. La preparazione sperimentale con una serie di punti equidistanti segnata da punti neri il cui ottica registrazioni può essere visto in (C). B. Esempio di tracciamento V m ed il gatto da una posizione centrale sulla matrice (vedi box in (C)). C. V m (blu) e CAT (rosso) dalla serie di punti equidistanti. Segnali sono stati cestinate 3x3. <img alt="Figura 4" src="/files/ftp_upload/3275/3275fig4.jpg" /> Figura 4. Mappa di attivazione e conduzione. R. Un esempio di attivazione mappa di un cuore selvaggio del mouse di tipo trasversale con (T) e longitudinale (L) le direzioni indicate dalle frecce bianche. B. V segnali m (in alto) e dV / dt (basso) corrispondenti ai tre punti di vista in A (T1, T2, T3, L1, L2, L3). <img alt="Figura 5" src="/files/ftp_upload/3275/3275fig5.jpg" /> Figura 5. Potenziale d&#39;azione e l&#39;analisi della durata di calcio transitori. Durata del potenziale d&#39;azione A. ripolarizzazione a 80% (APD80) e la durata transitoria calcio a 80% relax (CAD80) le mappe sono indicate da un cuore in condizioni di controllo (sinistra) e dopo 30 applicazioni nM isoproterenolo (Destra). Il colore giallo / verde nei ventricoli (a destra) indica isoproterenolo accorciato APD80 e CAD80. B. Esempio di tracciati APD80 e CAD80 dai ventricoli del mouse di tipo selvatico (Top) e atri (in basso).

<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>

Nessun conflitto di interessi dichiarati.

In questo esperimento abbiamo modificato il metodo di perfusione Langendorff con l&#39;aggiunta di un piccolo tubo di silicone, che è fondamentale soprattutto dopo la soppressione delle contrazioni del ventricolo con una eccitazione-contrazione disaccoppiatore. Il tubo di silicone viene usato per prevenire la congestione soluzione, l&#39;acidificazione della soluzione di perfusione, e lo sviluppo di ischemia del ventricolo sinistro. Il cuore del mouse è molto sensibile alla ipotermia, quindi, variazioni di temperatura in tutto il cuore artificiale a causa delle differenze durata del potenziale d&#39;azione. Di conseguenza, un sistema di riscaldamento è stato implementato nella camera di perfusione al fine di mantenere una temperatura costante di 37 ° C durante l&#39;intero dell&#39;esperimento 8. Dal momento che un modello Langendorff non conserva innervazione del cuore, bisogna considerare l&#39;aggiunta di neurotrasmettitori al perfusato al fine di indagare fisiologico tono simpatico e parasimpatico 9. Oltre perfusione retrograda, l&#39;aggiunta di superfusione del cuore aiuta a mantenere adeguati parametri ambientali quali pH e temperatura. In questo metodo, il cuore Langendorff perfuso era collocato orizzontalmente. Una verticale di impostazione perfusione Langendorff può essere utilizzato anche 10, ma può provocare meccanica cardiaca leggermente diverso 11. In aggiunta alle telecamere CMOS, rivelatori alternativa sono anche disponibili e possono essere applicati per mappare V m e Cat contemporaneamente 12. Applicazione delle telecamere CMOS ad alta risoluzione spazio-temporale garantisce l&#39;accuratezza delle registrazioni, tuttavia, i segnali ottici non sono la mappatura da una singola cellula. Piuttosto, ogni segnale fluorescente viene da centinaia o migliaia di cellule, a seconda di ingrandimento ottico. La fluorescenza ventricolare molto più grande può distorcere i segnali atriale di dispersione ottica, quindi, un&#39;attenta interpretazione dei segnali registrati otticamente è richiesto. Un altro limite della preparazione del mouse è la distorsione del segnale e rumore indotto dalla curvatura della superficie a causa delle ridotte dimensioni del cuore 13. Misure di velocità di conduzione può essere alterato non solo dalla curvatura del cuore del mouse ma anche dalla polarità degli elettrodi e gli elettrodi virtuale. Per ottenere una precisione di velocità di conduzione, anisotropia attivazione e le mappe della ripolarizzazione, corretta messa a fuoco della fotocamera sulla superficie del cuore è essenziale. In questo metodo, in tempo reale le registrazioni ECG possono integrare l&#39;indagine ottica di elettrofisiologia cardiaca. Voltaggio-sensibile colorante (RH237) e l&#39;indicatore di calcio (Rhod-2) sono utilizzati nel protocollo a causa della loro rapida risposta, eccitazione simili, e spettri di emissione distinti 3,7. Ci sono combinazioni alternative di coloranti che possono essere utilizzati per misurare V m e CAT diversi RH237 e Rhod-2:00 3. Un romanzo di tensione-sensibili colorante, PGHI, con un grande Stoke shift (&gt; 200 nm) è stato trovato per permettere una migliore m V e segnali gatto perché della separazione maggiore delle lunghezze d&#39;onda di emissione tra PGHI e Rhod-2:00 14. Futuri miglioramenti possono concentrarsi su esplorare nuove sonde fluorescenti, lo sviluppo di rivelatori di imaging, e migliorato il software di elaborazione delle immagini. Più alta risoluzione e nuove modalità di imaging ottico per la mappatura 3D ottica sono importanti anche le direzioni future della mappatura ottica 5.

<table border="1"><tbody><tr><td> Chimico </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> NaCl </td><td> Fisher Scientific, Fair Lawn, New Jersey </td><td> S271-1 </td></tr><tr><td> CaCl 2 (2H 2 O) </td><td> Fisher Scientific, Fair Lawn, New Jersey </td><td> C79-500 </td></tr><tr><td> KCl </td><td> Fisher Scientific, Fair Lawn, New Jersey </td><td> S217-500 </td></tr><tr><td> MgCl 2 (6H 2 O) </td><td> Fisher Scientific, Fair Lawn, New Jersey </td><td> M33-500 </td></tr><tr><td> NaH 2 PO 4 (H 2 O) </td><td> Fisher Scientific, Fair Lawn, New Jersey </td><td> S369-500 </td></tr><tr><td> NaHCO 3 </td><td> Fisher Scientific, Fair Lawn, New Jersey </td><td> S233-3 </td></tr><tr><td> D-glucosio </td><td> Fisher Scientific, Fair Lawn, New Jersey </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, 02:00 </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> Dimetilsolfossido (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

Il cuore del mouse è un modello importante per gli studi cardiovascolare a causa dell&#39;esistenza di tecnologie a basso costo per l&#39;ingegneria genetica in questa specie. Cardiovascolare fenotipizzazione fisiologica del cuore mouse può essere fatto facilmente utilizzando l&#39;imaging di fluorescenza utilizzando varie sonde per il potenziale transmembrana (V m), transienti di calcio (CAT), e altri parametri. Accoppiamento eccitazione-contrazione è caratterizzato da dinamiche di calcio intracellulare del potenziale d&#39;azione e, quindi, è di fondamentale importanza per mappare sia m V e CAT contemporaneamente dalla stessa posizione sul cuore 1-4. Simultanea mappatura ottica da Langendorff perfusi cuori mouse ha il potenziale per chiarire i meccanismi alla base dell&#39;insufficienza cardiaca, aritmie, malattie metaboliche, e di altre malattie cardiache. Visualizzazione di attivazione, velocità di conduzione, la durata del potenziale d&#39;azione, e altri parametri a una miriade di siti non possono essere raggiunti da indagini a livello cellulare, ma è ben risolto con la mappatura ottica 1,5,6. In questo articolo presentiamo l&#39;installazione della strumentazione e delle condizioni sperimentali per la mappatura simultanea ottica di V m e Cat nei cuori del mouse ad alta risoluzione spazio-temporale con state-of-the-art della tecnologia di imaging CMOS. Coerenti registrazioni ottiche ottenute con questo metodo che illustrano la mappatura simultanea ottico dei cuori del mouse Langendorff perfusi è fattibile e affidabile.

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

In questo documento i dettagli della procedura di dissezione, la configurazione strumentale, e delle condizioni sperimentali durante la mappatura ottica del potenziale transmembrana (Vm) e calcio intracellulare transitoria (CAT) in intatti Langendorff isolato perfuso cuori mouse.

Mappatura ottico dei potenziali d&#39;azione e transitori Calcio nel Cuore mouse

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

optical-mapping-action-potentials-calcium-transients-mouse

Research

JoVE Journal

Bioengineering

27.0K Views.  Washington University in St. Louis. In questo documento i dettagli della procedura di dissezione, la configurazione strumentale, e delle condizioni sperimentali durante la mappatura ottica del potenziale transmembrana (Vm) e calcio intracellulare transitoria (CAT) in intatti Langendorff isolato perfuso cuori mouse.

Video: 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.

Tridimensionale con risoluzione ottica Microscopia fotoacustico

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

Ricerca

Bioingegneria

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.

Multiparametrica mappatura ottica del Langendorff-perfuso Rabbit Heart

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.

Un Cre-Lox P approccio ricombinazione per la rilevazione di fusione cellulare 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.

In sintesi in vitro di Native, Spaziatura fibrosa lungo e segmentale collagene spaziatura lungo

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).

Rilevamento ottico di<em&gt; E. coli</em&gt; Batteri di mesoporosi 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 ...

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.

In Situ mappatura delle proprietà meccaniche del biofilm da-Particle tracciamento Microrheology

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.

Preparazione del 3D collagene Gel e microcanali per lo studio delle interazioni 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.

Microscopia di fluorescenza di luce-foglio di catturare immagini 4-dimensionale degli effetti di modulazione dello sforzo di taglio sul cuore Zebrafish in via di sviluppo

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.

Microscopia di fluorescenza di luce-foglio per lo studio del cuore murino

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.

Caratterizzazioni biassiale meccaniche delle valvole atrioventricolari cuore

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