Questo lavoro è stato sostenuto dalla Deutsche Forschungsgemeinschaft (KFO 196 &quot;Signaltransduktion bei adaptativen und maladaptiven kardialen rimodellante-Prozessen&quot;, FR 1638 / 1-2) e dalla DZHK (Centro tedesco per la ricerca cardiovascolare, una parte dei Centri tedeschi of Health Research , che è un BMBF (Ministero tedesco dell&#39;Istruzione e della Ricerca), iniziativa).

NOTA: I passaggi fondamentali della procedura di isolamento sono mostrati in Figura 1 Tutti i passaggi sono descritti in dettaglio nel seguente protocollo.. Isolamento del muscolo papillare, il montaggio in organo da camera bagno, acquisizione e l&#39;analisi viene effettuata in un lasso di tempo consecutivo e obbligatoria. Tutti gli esperimenti sugli animali sono stati eseguiti in conformità con la legislazione tedesca in materia di protezione degli animali e sono state approvate dal Consiglio di esame etico dell&#39;Università di Heidelberg. 1. Preparazione di Instrumentation <ol><li> Per contrattilità misurazioni utilizzare un setup vasca organo multichambered. Componenti di un ex vivo contrattilità configurazione bagno organo sono mostrati nella Figura 2. NOTA: Per minimizzare la quantità di tempo tra la preparazione del muscolo papillare e l&#39;inizio del protocollo sperimentale, impostare attrezzature e preparare buffer prima di raccogliere tissue.This sperimentali provides la maggior quantità di tempo che il tessuto rimane vitale per condurre esperimenti. </li><li> Avviare computer e apparecchiature di acquisizione dati. Attivare bagno organo di calore (preimpostato a 32 ° C) e lasciare unità alla temperatura preimpostata. </li><li> Preparare l&#39;apparecchiatura di acquisizione. Iniziare la registrazione dei dati come indicato nel manuale del software. </li><li> Controllare e condurre calibrazione (se necessario) su ciascun canale e azzerare tutti i canali di standardizzare il segnale elettrico fornito dal rispettivo trasduttore di forza (associato a quel canale) a 2 g forza fornita da un peso certificata. </li><li> Accendere approvvigionamento di gas (95% O 2/5% di CO 2) alle camere bagno organo. Continuamente gas tutte le camere durante l&#39;intero misure di protocollo. </li></ol> 2. Preparazione di Tamponi e soluzioni fisiologiche <ol><li> Preparare una soluzione tampone Krebs-Henseleit per raggiungere concentrazioni finali di 119 mM NaCl; 25 NaHCO MM 3; 4.6 mM KCl; 11MM glucosio; 1 mM Na-pyruvate; 1.5 mM CaCl 2; 1.64 mM MgSO 4; 1.18 mM KH 2 PO 4 e regolare il pH a 7,4 (vedi Tabella 1 soluzione fisiologica). </li><li> Preparare una soluzione tampone a parte Krebs-Henseleit da utilizzare durante l&#39;isolamento come descritto al punto 2.1 con l&#39;aggiunta di monoxime 30MM 2,3-butanedione (BDM) e 50 IE Eparina / ml. Raffreddare la soluzione a 4 ° C (vedere Tabella 1 Preparazione Soluzione). Per la simulazione ischemia, ischemia preparare soluzione come descritto nella Tabella 1. </li><li> Per il protocollo del Ca 2 + - contrattilità dipendente, aggiungere Ca 2+ alla soluzione tampone Krebs-Henseleit a concentrazione finale richiesta. NOTA: preparare queste soluzioni direttamente prima del suo utilizzo e Gass con CarboGen per evitare la precipitazione di Carbonato di Calcio. Soluzione Preriscaldare a 32 ° C prima dell&#39;uso. </li></ol> 3. Preparazione del Gassing Tube e dissezione del piatto utilizzata durante il Excision Procedure papillare <ol><li> Collegare un gas morbidocantare tubo (diametro esterno 2-4 mm) per il collegamento del gas (ossigeno al 100%, CarboGen alternativamente) con l&#39;esperto di lavoro. </li><li> Creare un anello chiuso del tubo gassazione raccordo all&#39;interno del piatto dissezione. Forare il tubo di gassificazione più volte che una ebollizione continua è assicurata. Questo tubo gassazione può essere utilizzato più volte. </li><li> Preparare piatto dissezione con un elastomero di silicone nella parte inferiore (0,5 cm), in modo che i perni possono essere bloccati in esso. Questo piatto può essere utilizzato più volte. </li></ol> 4. Isolamento dei anteriore sinistra Muscolo papillare da topi NOTA: Prima di iniziare l&#39;isolamento del muscolo papillare, controllare che le linee di gas sono chiare di blocchi. <ol><li> Preparare configurazione vasca organo come menzionato nella sezione del protocollo 1-3 prima di iniziare la preparazione dei muscoli papillari. Preparare tutte le soluzioni al giorno dell&#39;esperimento. </li><li> Sacrifica il mouse (circa 8-12 settimane) da cervicale dislocazione According per linee guida istituzionali di lavoro di esperti e. </li><li> Fissare l&#39;animale in posizione supina fissando zampe anteriori e posteriori zampe su una tavola di dissezione, aprire il torace laterali su entrambi i lati tagliando attraverso le costole con forbici ossee affilate, poi tagliare la membrana con un taglio trasversale. Rimuovere il pericardio. Ora cuore e l&#39;aorta sono accessibili. </li><li> Fissare il cuore con una pinza smussato sul tronco vascolare vicino al cuore e separare il cuore rapidamente con le forbici dai polmoni e il tessuto circostante. </li><li> Trasferire il cuore pulsante di una capsula di Petri riempite con una soluzione preparazione raffreddata gasati con l&#39;ossigeno (punto 2.2). Lasciare che il cuore a battere e stimolare le contrazioni del cuore dolcemente via toccando l&#39;apice cardiaco con una pinza. </li><li> Non appena il cuore è completamente exsanguinous, trasferirlo al piatto dissezione riempito con soluzione di preparazione raffreddata (passo 2.2) e gasati con O 2. Da questo punto in uso uno stereomicroscopio. </li><li> Fissare il heaRT con un piccolo perno attraverso il ventricolo destro in modo che il cuore è visto dalla dorsale (ventricolo destro si trova sul lato destro dalla vista dell&#39;operatore). </li><li> Utilizzando le forbici microchirurgiche separano entrambi gli atri a livello atrioventricolare, taglio tessuto connettivo dai ventricoli e scartare. Effettuare un taglio attraverso la parete ventricolare dalla valvola livello AV all&#39;apice del cuore evitando ogni pressione meccanica. </li><li> Aprire il ventricolo e fissare la parete libera lato sinistro con una pinza, avendo così uno sguardo a due ventricolo muscoli papillari sinistro. </li><li> Leggermente tagliare il tessuto ventricolare laterali su entrambi i lati del muscolo papillare anteriore sinistra conservando una parte della vela valvolare sulla preparazione muscolo papillare ed evitare di toccare o allungamento del muscolo papillare quanto possibile. Sezionare il restante tessuto della parete ventricolare dal muscolo papillare. </li><li> Fissare fili di seta (7/0, metrico 0,5) su entrambi i lati della preparazione muscolo papillarearazione, uno sulla vela valvolare ed una sulla parte muscolare. Fissare la preparazione nella camera bagno di organo pieno di soluzione del bagno organo e gasati con CarboGen, la temperatura a 32 ° C viene consigliata. </li></ol> 5. equilibrazione e stimolazione del muscolo papillare <ol><li> Stimolare la preparazione muscolo papillare con impulsi rettangolari (durata di 2 ms e corrente di 100 mA) a frequenza di 1 Hz stimolazione immediatamente dopo il fissaggio nel bagno organo. </li><li> Assicurare un frequente ricambio della soluzione del bagno di organo, sia dal cambiamento continuo o da frequenti modifica manuale (ogni 5 min) a seconda dell&#39;impostazione del tipo a bagno d&#39;organo utilizzato. </li><li> Aumentare gradualmente la pretesa fino a forza contrazione massima a raggiunto. Forza contrazione massima viene raggiunta quando l&#39;aumento del precarico non è seguito da un ulteriore aumento nella forza contrazione. </li><li> Dopo 45-60 minuti di equilibrio, avviare il protocollo sperimentale. </li></ol> NOTA: Il protocollo sperimentale descritto di seguito comprende manovre standard per caratterizzare la contrattilità cardiaca in condizioni fisiologiche e fisiopatologiche. Nella sezione rappresentante descriviamo questi protocolli in dettaglio anche mostrando i risultati di rappresentanza (vedi anche tabella 2). <ol><li> Registrazione ed analisi dei protocolli sperimentali <ol><li> Per la registrazione, utilizzare un software adatto con adeguata risoluzione temporale (velocità di acquisizione ≥ 1kHz). </li><li> Analizzare parametri tra cui la forza contrazione ampiezza (altezza), il tempo di picco di tensione (TTP) e per metà tempo di rilassamento massimo (R 50 / tfall, rispettivamente) come indicato nel programma di software (come ad esempio Grafico 5.5 di ADInstruments, vedi Figura 4). </li></ol></li></ol>

<ol>
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Ca(2+)-dependent and Ca(2+)-independent regulation of contractility in isolated human myocardium.</a> Basic Res Cardiol. 92, 75-86 (1997).</li><li>Corbin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sildenafilcitrate+does+not+affect+cardiac+contractility+in+human+or+dog+heart.">Sildenafilcitrate does not affect cardiac contractility in human or dog heart.</a> Curr Med ResOpin. 19 (8), 747-752 (2003).</li><li>Romero-Vecchione, E., Vasquez, J., Rosa, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+negative+inotropic+effect+of+cocaine+in+rat+ventricle+strip.">Direct negative inotropic effect of cocaine in rat ventricle strip.</a> Acta Cient Venez. 47 (1), 17-23 (1996).</li><li>N&#228;bauer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+inotropic+effects+in+isolated+ventricular+myocardium+from+nonfailing+and+terminally+failing+human+hearts.">Positive inotropic effects in isolated ventricular myocardium from nonfailing and terminally failing human hearts.</a> Eur J Clin Invest. 18 (6), 600-606 (1988).</li><li>Gao, W. 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G., Jewell, B. R., Wood, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+the+contractility+of+mammalian+myocardium+at+low+rates+of+stimulation.">Studies of the contractility of mammalian myocardium at low rates of stimulation.</a> J Physiol. 254 (1), 1-17 (1976).</li><li>Pieske, B., Maier, L. S., Schmidt-Schweda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sarcoplasmic+reticulum+Ca2++load+in+human+heart+failure.">Sarcoplasmic reticulum Ca2+ load in human heart failure.</a> Basic Res Cardiol. 97, 163-171 (2002).</li><li>Koch-Weser, J., Blinks, J. 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+Influence+of+the+Interval+between+Beats+on+Myocardial+Contractility.">The Influence of the Interval between Beats on Myocardial Contractility.</a> Pharmacol Rev. 15, 601-652 (1963).</li><li>Bocalini, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+remodeling+after+large+infarcts+in+rat+converts+post+rest-potentiation+in+force+decay.">Myocardial remodeling after large infarcts in rat converts post rest-potentiation in force decay.</a> Arq Bras Cardiol. 98 (3), 243-251 (2012).</li><li>Juggi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ischemia-reperfusion+on+the+post-rest+inotropy+of+isolated+perfused+rat+heart.">Effect of ischemia-reperfusion on the post-rest inotropy of isolated perfused rat heart.</a> J Cell Mol Med. 6 (4), 621-630 (2002).</li><li>Lakatta, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+Bowditch:+the+convergence+of+cardiac+chronotropy+and+inotropy.">Beyond Bowditch: the convergence of cardiac chronotropy and inotropy.</a> Cell Calcium. 35 (6), 629-624 (2004).</li><li>Taylor, D. G., Parilak, L. D., LeWinter, M. M., Knot, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+the+rat+left+ventricle+force+and+Ca2++-frequency+relationships:+similarities+to+dog+and+human.">Quantification of the rat left ventricle force and Ca2+ -frequency relationships: similarities to dog and human.</a> Cardiovasc Res. 61 (1), 77-86 (2004).</li><li>Schmidt, U., Hajjar, R. J., Gwathmey, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+force-interval+relationship+in+human+myocardium.">The force-interval relationship in human myocardium.</a> J Card Fail. 1 (4), 311-321 (1995).</li><li>Rossman, E. I., Petre, R. E., Chaudhary, K. W., Piacentino, V. 3. r. d., Janssen, P. M., Gaughan, J. P., Houser, S. R., Margulies, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+frequency-dependentresponses+represent+the+pathophysiologic+signature+of+contractile+failure+inhuman+myocardium.">Abnormal frequency-dependentresponses represent the pathophysiologic signature of contractile failure inhuman myocardium.</a> JMCC. 36 (1), 33-42 (2004).</li><li>Moran, A. E., Forouzanfar, M. H., Roth, G. A., Mensah, G. A., Ezzati, M., Murray, C. J., Naghavi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+trends+in+ischemic+heart+disease+mortality+in+21+world+regions,+1980+to+2010:+the+Global+Burden+of+Disease+2010+stud.">Temporal trends in ischemic heart disease mortality in 21 world regions, 1980 to 2010: the Global Burden of Disease 2010 stud.</a> Circulation. 129 (14), 1483-1492 (1980).</li><li>Lee, J. A., Allen, D. 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Gli autori dichiarano di non avere interessi finanziari in competizione.

In questo manoscritto si descrive un metodo per indagare la contrattilità del muscolo papillare murino in vitro che può essere utilizzato per rispondere ad alcune domande scientifici relativi alla fisiologia del cuore e la patologia nei topi, nonché per sostenere l&#39;analisi delle linee transgeniche e la scoperta di nuovi approcci farmaceutici per il trattamento di disfunzioni cardiache. Illustriamo l&#39;uso di questo metodo per valutare le proprietà fisiologiche, patologiche e farmacologiche di contratt...

L&#39;indagine di proteine ​​come canali ionici che si riferiscono loro ruolo per la contrattilità cardiaca è essenziale per scoprire differenti meccanismi patogenetici e di stabilire nuove strategie terapeutiche per le malattie cardiache come l&#39;ischemia e insufficienza cardiaca. Funzione contrattile dei cardiomiociti mammiferi è noto per essere modulata da vari canali ionici, trasportatori e altre proteine. Potenziale d&#39;azione evocato attivazione di tensione dipendente sarcolemmal tipo L di Ca 2+ canali porta a Ca 2+ afflusso dallo spazio extracellulare e successivamente Ca2 + indotta rilascio di Ca 2+ (CICR) 1, che innesca la contrazione cellulare 2. Ca 2+ -Segnalazione gioca un ruolo centrale nella contrattilità cardiaca e l&#39;adattamento allo stress fisiologico o patologico. Catecolamine attivano i recettori beta-adrenergici cardiaci, stimolando così ciclasi (AC) che sintetizza cAMP. L&#39;attivazione, Protein chinasi A (PKA) fosforila diverse proteine ​​associate intracellulari e di membrana come L-tipo Ca 2+ canali, fosfolambano e recettori rianodinici conseguente modifica di Ca 2+ transitori e cardiaco contrattilità 1,3,4. cAMP è degradata dalla fosfodiesterasi (PDE). L&#39;attivazione dei recettori Gs-accoppiati diversi beta-adrenergici porta anche ad accumulo di cAMP. La tecnica di misurazione contrattilità in isolati strisce muscolari ventricolare è ben definito per le più grandi mammiferi 5-8. Sulla base della possibilità di gene targeting in topi è importante stabilire metodi per analizzare murino fisiologia cardiaca. Tuttavia, i dati esistenti sulle proprietà fisiologiche di preparati muscolari isolate in topi variano a seconda delle condizioni sperimentali 9-12. Il metodo descritto è utilizzato per analizzare contrattilità cardiaca ventricolare sinistra pre muscolo papillareparations in vitro. Investigation della contrattilità cardiaca viene eseguita in assenza di influenze modificanti contrattilità cardiaca in vivo, come la pressione sanguigna, stimolazione neuroumorale e stress fisico o metabolica. Il tasso di battito della preparazione contraente muscolo può essere rigorosamente definito e cambiato arbitrariamente. Twitch forza può essere analizzato nel contesto di stimoli specifici come concentrazione di calcio, battendo frequenza o temperatura. Inoltre, questo metodo può essere usato per studiare diversi componenti della via di segnalazione e di confrontare funzione cardiaca di modelli murini geneticamente modificati controllando condizioni sperimentali sopra menzionati.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5761&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>D9434&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P5280&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td align="right">223506</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate</td>
			<td>Sigma-Aldrich</td>
			<td align="right">230391</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>P 5655</td>
			<td></td>
		</tr>
		<tr>
			<td>2,3-Butanedione monoxime</td>
			<td>Sigma-Aldrich</td>
			<td>B0753</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma-Aldrich</td>
			<td>F3917</td>
			<td>Hazard statement H312, solve in DMSO</td>
		</tr>
		<tr>
			<td>3-?Isobutyl-?1-?methylxanthine</td>
			<td>Sigma-Aldrich</td>
			<td>I5879</td>
			<td>Hazard statement H 302, solve in DMSO</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoprenaline hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>I5627</td>
			<td>Hazard statement H 315-H319-H335</td>
		</tr>
		<tr>
			<td>Sodium Heparine 250.000 IE/10ml</td>
			<td>ratiopharm</td>
			<td>PZN 3874685</td>
			<td></td>
		</tr>
		<tr>
			<td>Histamine dihydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>H7250</td>
			<td>Hazard statement H 315-H 317-H319- H334-H335</td>
		</tr>
	</tbody>
</table>

contractility measurements on isolated papillary muscles for the investigation of cardiac inotropy in mice

Muscoli papillari isolati dai cuori adulte di topo possono essere utilizzati per studiare la contrattilità cardiaca durante le diverse condizioni patologiche / fisiologiche. Le caratteristiche contrattili possono essere valutate in maniera indipendente da influenze esterne, quali tono vascolare o lo stato neuroumorale. Raffigura un approccio scientifico tra le misurazioni di cellule singole con isolate miociti cardiaci e studi in vivo, come l&#39;ecocardiografia. Così, preparazioni muscolo papillare servire come un modello eccellente per studiare la fisiologia cardiaca / patofisiologia e può essere utilizzato per le indagini come la modulazione da agenti farmacologici o l&#39;esplorazione di modelli animali transgenici. Qui, descriviamo un metodo per isolare l&#39;anteriore sinistra muscolo papillare murino di indagare la contrattilità cardiaca in un setup bagno di organo. A differenza di un preparato striscia muscolare isolato dalla parete ventricolare, il muscolo papillare può essere preparato in toto senza danneggiare il muscolo tissuuna e severamente. Il setup bagno organo è composto da diversi elettrodi attrezzate camere a bagno d&#39;organo a temperatura controllata, gasati e. Il muscolo papillare isolato è fissato nella camera bagno di organo e stimolato elettricamente. La forza di contrazione evocata viene registrata utilizzando un trasduttore di pressione e parametri quali contrazione forza ampiezza e contraggono la cinetica vengono analizzati. Diversi protocolli sperimentali possono essere eseguite per indagare la contrattilità e calcio-dipendente dalla frequenza e curve dose-risposta di agenti contrattili come catecolamine o altri farmaci. Inoltre, le condizioni patologiche come l&#39;ischemia acuta possono essere simulati.

Il protocollo di questo manoscritto per misure contrattilità isolati murini preparazioni muscolo papillare è sintonizzato per condizioni ottimali per ottenere risultati sperimentali riproducibili in condizioni fisiologiche. Per definire le condizioni sperimentali ottimali abbiamo eseguito esperimenti pilota variabili temperatura del bagno d&#39;organo e la concentrazione di calcio extracellulare (vedi anche 12). Il protocollo qui descritto è stato eseguito con una concentrazione di calcio extracellulare 1,...

Watch this Scientific Journal Video about Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice at JoVE.com

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Murine sinistra muscolo papillare ventricolare può essere usato per studiare la contrattilità cardiaca in vitro. Questo articolo descrive in dettaglio l&#39;isolamento e protocolli sperimentali per studiare le caratteristiche contrattili cardiaci.

Misure contrattilità isolati su muscoli papillari per le inchieste sui Cardiac inotropo nei topi

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

contractility-measurements-on-isolated-papillary-muscles-for

Research

JoVE Journal

Biology

14.9K Views.  Universität Heidelberg.  Murine sinistra muscolo papillare ventricolare può essere usato per studiare la contrattilità cardiaca in vitro. Questo articolo descrive in dettaglio l'isolamento e protocolli sperimentali per studiare le caratteristiche contrattili cardiaci. 

Video: Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

Patch clamp e Tecniche di perfusione per la valutazione dei canali ioni Espresso in Xenopus Ovociti

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Ricerca

Biologia

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

Bioingegneria reti umane in topi immunodeficienti microvascolari

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables. 
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

Il Ex vivo Preparazione isolato microvasi scheletrico per le Indagini di reattività vascolare

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

Vivo Valutazione Ex della contrattilità, affaticabilità e alternanza in isolati muscoli scheletrici

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

Microscopia intravitale del microcircolo nel muscolo mouse Cremaster per l&#39;analisi di migrazione delle cellule staminali periferiche

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

La tecnica del rene perfusi mouse isolato

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

Analisi del trasferimento di gig Junction-dependent di miRNA con la microscopia 3D-FRAP

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. In vivo cardiac-specific gene delivery is commonly achieved by either systemic or local delivery. Systemic injection via tail vein is easy and efficient in manipulating cardiac gene expression by using recombinant adeno-associated virus 9 (AAV9). However, this method requires a relatively high amount of vector for efficient transduction, and may result in nontarget organ gene transduction. Here, we describe a simple, efficient, and time-saving method of intramyocardial injection for in vivo cardiac-specific gene manipulation in mice. Under anesthesia (without ventilation), the pectoral major and minor muscles were bluntly dissected, and the mouse heart was quickly exposed by manual externalization through a small incision at the fourth intercostal space. Subsequently, adenovirus encoding luciferase (Luc) and vitamin D receptor (VDR), or short hairpin RNA (shRNA) targeting VDR, was injected with a Hamilton syringe into the myocardium. Subsequent in vivo imaging demonstrated that luciferase was successfully overexpressed specifically in the heart. Moreover, Western blot analysis confirmed the successful overexpression or silencing of VDR in the mouse heart. Once mastered, this technique can be used for gene manipulation, as well as injection of cells or other materials such as nanogels in the mouse heart.

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Here we present a protocol for cardiac-specific gene manipulation in mice. Under anesthesia, the mouse hearts were externalized through the fourth intercostal space. Subsequently, adenoviruses encoding specific genes were injected with a syringe into the myocardium, followed by protein expression measurement via in vivo imaging and Western blot analysis.

Un metodo semplice ed efficace per In Vivo manipolazione del Gene specifico tramite l'iniezione intramiocardica in topi

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...

Analyzing the &alpha;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. Despite the development of multiple differentiation protocols, the generation of iPSC cardiomyocytes resembling an adult-like phenotype remains challenging. One major aspect of cardiomyocytes maturation involves the formation of a well-organized sarcomere network to ensure high contraction capacity. Here, we present a super resolution-based approach for semi-quantitative analysis of the &#945;-actinin network in cardiomyocytes. Using photoactivated localization microscopy a comparison of sarcomere length and z-disc thickness of iPSC-derived cardiomyocytes and cardiac cells isolated from neonatal tissue was performed. At the same time, we demonstrate the importance of proper imaging conditions to obtain reliable data. Our results show that this method is suitable to quantitatively monitor the structural maturity of cardiac cells with high spatial resolution, enabling the detection of even subtle changes of sarcomere organization.

Analyzing the &#945;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The formation of a proper sarcomere network is important for the maturation of iPSC-derived cardiomyocytes. We present a super resolution-based approach that allows for the quantitative evaluation of the structural maturation of stem cell derived cardiomyocytes, to improve culture conditions promoting cardiac development.

Analisi della α-Actinin Network in Cardiomiociti derivati da iPSC umani utilizzando la microscopia di localizzazione a singola molecola

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. ...

Dissection of the Endolymphatic Sac from Mice

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the human phenotypes. Although important progress has been done in the understanding of the development and function of the neurosensory epithelia of the cochlea and vestibula, limited knowledge is available regarding the development, cellular composition, molecular pathways and functional characteristics of the endolymphatic sac. This is, in large part, due to the difficulty of visualizing and microdissecting this tissue, which is an epithelium comprised of only one cell layer. The study presented here describes an approach to access and microdissect the endolymphatic sac from the wild-type mouse inner ear at different ages. The result of a similar dissection is shown in a pendrin-deficient mouse model of enlargement of the vestibular aqueduct. A transgenic mouse with a fluorescent endolymphatic sac is presented. This reporter mouse can be used to readily visualize the endolymphatic sac with limited dissection and determine its size. It can also be used as an educational tool to teach how to dissect the endolymphatic sac. These dissection procedures should facilitate further characterization of this understudied part of the inner ear.

<meta charset="utf8"></head><body>Dissezione del sacco endolinfatico da topi

This study describes how to dissect the endolymphatic sac from the inner ear of the mouse at different ages. The result of a similar dissection is shown in an Slc26a4-null mouse model of enlargement of the endolymphatic sac. A transgenic mouse with a fluorescent reporter expressed in the endolymphatic sac is presented as a model to readily visualize the endolymphatic sac, and educational tool.

Dissezione del sacco endolinfatico da topi

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the ...