Inoltre, ringraziamo i cardiochirurghi dell&#39;Università di Heidelberg per la fornitura di tessuto atriale umano e Claudia Liebetrau, Katrin Kupser e Ramona Nagel per l&#39;eccellente supporto tecnico. Un ringraziamento speciale anche a Andy W. Trafford per i suoi utili suggerimenti e consigli durante la creazione dei Ca 2 + misure transitorie. Gli autori desiderano esprimere la loro gratitudine più profonda per i membri del Dipartimento di Farmacologia e Tossicologia (testa: Ursula Corvi) del Dresden University of Technology per l&#39;opportunità che ci hanno offerto di apprendere le tecniche di base e le competenze in elettrofisiologia cellulare e l&#39;isolamento dei cardiomiociti. Ricerca degli autori è sostenuta dal tedesco Research Foundation (Do769/1-1-3), il ministero federale tedesco dell&#39;Istruzione e della ricerca attraverso la Fibrillazione Atriale Rete di competenza (01Gi0204) e il Centro tedesco per la ricerca cardiovascolare, l&#39;Unione europea attraverso l&#39;EuRete pea per la ricerca traslazionale nella fibrillazione atriale (EUTRAF, FP7-HEALTH-2010, progetto di integrazione di grandi dimensioni, Proposta N. 261057) e l&#39;europeo-nordamericano Atrial Fibrillation Research Alliance (ENAFRA) concessione di Fondation Leducq (07CVD03). La panoramica schematica mostrata nel file video è stato prodotto usando Servier arte medica.

Protocolli sperimentali devono essere approvati dal comitato etico locale e tutti i pazienti hanno bisogno di dare un consenso informato scritto. La nostra ricerca è stato approvato dal Comitato Etico della Facoltà di Medicina di Mannheim, Università di Heidelberg (# 2011-216n-MA) ed è stata eseguita in conformità a tutte le linee guida istituzionali, nazionali e internazionali per il benessere umano. Tutti i pazienti hanno dato consenso informato scritto. 0. Ottenere umana tessuto atriale Durante le procedure di incannulazione routine in pazienti sottoposti a chirurgia a cuore aperto per bypass cardiopolmonare innesto la punta del diritto appendice atriale è solitamente rimosso e può essere utilizzato per l&#39;isolamento di cardiomiociti atriali. Dopo l&#39;asportazione del campione di tessuto viene trasferito immediatamente in un tubo da 50 ml Falcon con sterile Ca 2 + senza soluzione di trasporto contenente 2,3-butanedione monoxime (tabella I, BDM, inibitore contrattile, impedendo miociti contracture). Con i tempi di trasporto tra i 30 ei 45 minuti, non abbiamo riconosciamo un chiaro vantaggio di raffreddamento o di ossigenazione per quanto riguarda sia il numero e la qualità dei isolate cardiomiociti atriali. In generale, il trasporto al laboratorio deve essere il più velocemente possibile. Tuttavia, se tragitto lungo, non può essere evitato, trasporto a 4 ° C in soluzione ossigenata potrebbe essere vantaggiosa. 1. Predisposizioni <ol><li> Accendere il thermocirculator, che controlla la temperatura del becher incamiciata (Tabella II). Assicurarsi che la temperatura all&#39;interno del bicchiere equivale a 37 ° C. Coprire il becher con un coperchio di vetro per mantenere una temperatura costante all&#39;interno del becher in tutto il processo di isolamento. </li><li> Pesare il Collagenasi I e proteasi XXIV in tre bicchieri di vetro e conservarla a temperatura ambiente. Gli enzimi saranno diluiti prima dell&#39;uso. </li><li> Conservare 20 ml di Ca 2 + soluzione gratuita in una capsula di Petri a 4 ° C. </li><li>Negozio 250 ml di Ca 2 + soluzione libera in un bagno d&#39;acqua a 37 ° C. </li></ol> 2. Pulizia del Tissue <ol><li> Trasferire il campione di tessuto insieme alla soluzione di trasporto in una capsula Petri (~ 10 cm di diametro) e rimuovere il tessuto adiposo all&#39;incirca usando forbici. </li><li> Pesare il campione di tessuto. Tra 200 e 600 mg sono utilizzati per l&#39;isolamento delle cellule. Il restante tessuto può essere congelato in azoto liquido per la successiva analisi biochimica. </li><li> Trasferire il campione di tessuto nella capsula di Petri contenente 20 ml di Ca 2 + soluzione a 4 ° C (vedi punto 1.3) e tritare il campione di tessuto in piccoli pezzi di circa 1 mm 3 di dimensione. </li><li> Le seguenti operazioni devono essere eseguite a 37 ° C e sotto la continua emissione di gas con il 100% O 2. Una punta di pipetta può essere posizionato sul tubo ossigeno ad evitare forti bubbling e generare il flusso d&#39;aria continuo. </li><li> Trasferire i pezzi di tessuto insieme alla soluzione 2 +-libero in Caper il bicchiere con guaina e mescolate con cura per circa 3 minuti con un agitatore magnetico. </li><li> Smettere di mescolare e lasciare i pezzi di tessuto di stabilirsi per qualche secondo. Colare attenzione il surnatante con una rete di nylon (200 micron, tabella II). Restituire i pezzi di tessuto trattenuto dalla maglia nel bicchiere con pinze. </li><li> Riempire il bicchiere con Ca 2 + soluzione gratuita e ripetere la fase di lavaggio 2.4 e 2.5 due volte. </li></ol> 3. Prima digestione enzimatica <ol><li> Risospendere i pezzi di tessuto con 20 ml di soluzione enzimatica E1 (Tabella I) contenente collagenasi e proteasi e mescolate con cura per 10 min. </li><li> Aggiungere 40 ml di 10 mM CaCl 2-soluzione per ottenere una concentrazione finale di 20 mM Ca 2 +. </li><li> Dopo 35 min forzerebbe il surnatante con cura attraverso una maglia di nylon (200 micron, tabella II) in un modo che la maggior parte dei pezzi di tessuto rimangono nel becher. Resto Ritornopiovuto pezzi di tessuto dalla maglia nel bicchiere. </li></ol> 4. Seconda digestione enzimatica <ol><li> Sospendere di nuovo i pezzi di tessuto con 20 ml di soluzione di enzima E2 contenente collagenasi ho solo (Tabella I). Aggiungere 40 ml di 10 mM CaCl 2-soluzione immediata per ottenere una concentrazione finale di 20 mM Ca 2 +. </li><li> Durante il secondo uso forbici digestione di tagliare ulteriormente i coaguli di tessuto di tanto in tanto. </li><li> Dopo 5 min prendere un campione con una pipetta Pasteur controlla la dissociazione delle cellule. Ripetere questa operazione ogni 2-3 minuti fino a quando appaiono a forma di bastoncello, cardiomiociti striati. </li><li> Smettere di mescolare e lasciare i pezzi di tessuto di stabilirsi per circa 20-30 sec. </li><li> Filtrare il surnatante con cura attraverso una rete di nylon (200 micron, tabella II) in una provetta da 50 ml Falcon (metro A). Restituire i pezzi di tessuto trattenuto dalla maglia nel bicchiere. </li><li> Risospendere i pezzi di tessuto nel bicchiere con 20 ml Storage soluzione (Tabella I) 10 e di dissociare ulteriormente le cellule dolce triturazione meccanica utilizzando un 20 ml pipetta sierologica con dispenser. Cercate di evitare la formazione di bolle in quanto le bolle sono dannose per la sopravvivenza delle cellule e di qualità. </li><li> Filtrare il surnatante con cura attraverso una rete di nylon (200 micron, la tabella II) in un tubo ml Falcon 50 (metro B). </li></ol> 5. Preparazione finale e regolazione di Finale Ca 2 + Concentrazione <ol><li> Centrifugare sia Falcon tubi A e B (vedi punto 4.5 e 4.7) a 95 g per 10 min. </li><li> Rimuovere il surnatante da entrambi i tubi con attenzione per aspirazione. Fare attenzione a non disturbare il pellet. Scartare il surnatante. </li><li> Risospendere pellet in entrambe le soluzione di storage da 1,5 ml (Tabella I) ogni (temperatura ambiente). </li><li> Aggiungere due volte 7,5 ml di 10 mM CaCl 2 soluzione per ogni Falcon e mescolate con cura. Incubare per 10 minuti dopo ogni passaggio. </li><li>Aggiungere 15 ml di 10 mM di soluzione di CaCl 2 per ottenere una finale Ca 2 + concentrazione di 0,2 mM. </li></ol> 6. Caricamento dei miociti con lo fluorescente Ca 2 +-Indicatore Fluo-3 AM (Figura 1) <ol><li> Trasferire 1,5 ml di sospensione cellulare (tubo A e / o il tubo B) in una provetta per microcentrifuga da 2 ml (provetta Eppendorf). </li><li> Le seguenti operazioni devono essere eseguite e considerando la sensibilità alla luce del Ca 2 + indicatore fluorescente Fluo-3. </li><li> Sciogliere 50 mg della membrana permeabile derivato estere acetossimetilico di Fluo-3 (Fluo-3 AM, Tabella III) in 44 microlitri della Pluronic F-127 (Tabella III) soluzione madre (20% w / v in DMSO anidro) per ottenere ± 1 mm Fluo-03:00 soluzione madre, che può essere conservato a -20 ° C per un massimo di 1 settimana. </li><li> Aggiungere 15 microlitri della Fluo-03:00 soluzione madre alla provetta contenente 1,5 ml di sospensione cellulare (vedi punto 6.1) e agitareattentamente. </li><li> Incubare la sospensione cellulare per 10 min in una scatola otticamente opaca. </li><li> Centrifugare brevemente a circa 6.000 giri al minuto. </li><li> Eliminare il supernatante e risospendere il pellet in 1,5 ml di soluzione del bagno (Tabella IV). </li><li> Lasciare la sospensione cellulare per circa 30 min per de-esterificazione prima di iniziare con gli esperimenti. </li></ol> 7. Simultanei patch-clamp e epifluorescente Ca 2 + Misure Dal momento che le misurazioni di patch-clamp non sono l&#39;argomento principale di questa recensione, rimandiamo il lettore interessato ad altre pubblicazioni che forniscono una descrizione più approfondita di questa tecnica. 11-14 Per completezza forniamo una breve sintesi di un protocollo per misurare potenziali d&#39;azione o di tipo L Ca 2 + correnti, entrambi con simultanei Ca 2 +-registrazioni transitori. Durante gli esperimenti miociti sono superfuse a 37 ° C con bagno solutisu (tabella IV) utilizzando un sistema di perfusione rapido (Octaflow IITM, ALA Scientific Instruments, NY). Per gli esperimenti di tensione-clamp, K + correnti vengono bloccati aggiungendo 4-aminopiridina (5 mmol / L) e BaCl 2 (0,1 mmol / L) alla soluzione del bagno. Microelettrodi di vetro borosilicato sono utilizzati e dovrebbero avere la mancia resistenze di 2-5 MW, quando riempita con pipetta soluzione (Tabella V). Oltre al Fluo-03:00 caricando dei miociti (vedere fase 6), Fluo-3 è anche incluso nella soluzione pipetta (Tabella V). Fluorescenza è eccitato a 488 nm e la luce emessa (&lt;520 nm) convertito [Ca 2 +] i supponendo <img alt="Equazione 1" fo:content-width="1.540in" fo:src="/files/ftp_upload/50235/50235eq1highres.jpg" src="/files/ftp_upload/50235/50235eq1.jpg" /> dove k d = costante di dissociazione di Fluo-3 (n 864M), F = Fluo-3 fluorescenza. F max = Ca 2 +-fluorescenza saturo ottenuto al termine di ogni esperimento 12 Entrambi i segnali elettrici e epifluorescente Ca 2 + segnali vengono registrate contemporaneamente. I potenziali d&#39;azione sono stimolate a 0,5 Hz in modalità current-clamp con 1 msec attuali impulsi di forza soglia 1.2x. L-tipo Ca 2 +-correnti sono misurati in modalità voltage-clamp utilizzando un potenziale di mantenimento di -80 mV e 100 msec-ramp-impulso a -40 mV per inattivare la rapida Na +-corrente, seguito da un 100-msec test-impulso a +10 mV a 0,5 Hz.

<ol>
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Res. 66, 1277-1286 (1990).</li><li>Hove-Madsen, L., 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=Atrial+fibrillation+is+associated+with+increased+spontaneous+calcium+release+from+the+sarcoplasmic+reticulum+in+human+atrial+myocytes.">Atrial fibrillation is associated with increased spontaneous calcium release from the sarcoplasmic reticulum in human atrial myocytes.</a> Circulation. 110, 1358-1363 (2004).</li><li>Molina, C. E., 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=Cyclic+adenosine+monophosphate+phosphodiesterase+type+4+protects+against+atrial+arrhythmias.">Cyclic adenosine monophosphate phosphodiesterase type 4 protects against atrial arrhythmias.</a> J. Am. Coll. Cardiol. 59, 2182-2190 (2012).</li></ol>

Worthington Biochemical Corp. sostenuto questa pubblicazione per coprire i costi di produzione di file video. I finanziatori non aveva alcun ruolo nel disegno dello studio, la raccolta e l&#39;analisi dei dati, la decisione di pubblicare, o preparazione del manoscritto.

Qui si descrive un metodo per l&#39;isolamento di miociti atriali umane da appendici atriali giuste ottenute da pazienti sottoposti a chirurgia a cuore aperto. Per poter utilizzare questi miociti per misure di Ca 2 + citosolico abbiamo adattato un metodo precedentemente descritto 4-11 omettendo dell&#39;EGTA dalla soluzione di storage. Già nel 1970 si è osservato che, sebbene miociti dissociano in presenza di Ca 2 + durante la digestione, tutti erano in contrattura e non vitali. 16,17 Pertanto, isolamento cellulare è eseguita in Ca 2 +-soluzione libera. Tuttavia, la reintroduzione di concentrazioni fisiologiche di Ca 2 + comportato rapida Ca 2 + afflusso e la morte cellulare. Questo è stato descritto come il Ca 2 + fenomeno paradosso originariamente osservata nei cuori perfusi da Zimmerman e Hulsman. 18 Le modifiche dei terreni di isolamento tra cui la riduzione del pH a 7,0, &lt;sup&gt; 19 aggiunta di taurina 20 o di piccole quantità di Ca 2 + (vedi punto 3.2 e 4.1), 21 come pure la memorizzazione dei miociti isolati in dell&#39;EGTA contenenti storage-soluzione 22 sono state proposte per evitare che il Ca 2 + paradosso. 17 Tuttavia, è ben noto che Ca 2 + attraverso bufferizzazione EGTA riduce l&#39;ampiezza di tipo L Ca 2 + corrente indotta Ca 2 + transitori ampiezze e comporta un decadimento delle bifasica Ca 2 + transitori. 23 Pertanto, abbiamo omesso EGTA durante l&#39;intero processo di isolamento per ottenere Ca 2 + transitori con proprietà tipiche e decadimenti monofasici. Per proteggere le cellule dal Ca 2 + paradosso abbiamo aumentato la finale Ca 2 + concentrazione della soluzione di stoccaggio in modo graduale fino a 0,2 mM. La scelta di collagenasi è probabilmente la fase più critica per il successo isolamento dei miociti. Coll convenzionaleagenases sono preparazioni grezze ottenute da Clostridium histolyticum e contengono collagenasi oltre ad una serie di altre proteinasi, polysaccharidases e lipasi. Sulla base delle loro collagenasi generali di composizione sono suddivise in diverse tipologie. Worthington 24 Tipi collagenasi I e II sono stati utilizzati con successo per l&#39;isolamento dei miociti atriali umani. 4-10,15,25-30 Nel nostro protocollo attualmente descritto si consiglia l&#39;uso di collagenasi tipo I, anche se siamo stati anche in grado di ottenere quantità accettabili di cellule vitali mediante collagenasi di tipo II. Tuttavia, anche all&#39;interno di un unico tipo collagenasi vi è una significativa variazione da lotto a lotto sulle attività enzimatiche. Queste variazioni richiedono un&#39;attenta selezione del lotto e la sperimentazione di vari lotti per ottimizzare la procedura di isolamento. Lo strumento a disposizione batch selezione on-line da Worthington Biochemical Corp. ( <a href="http://www.worthington-biochem.com/cls/match.php" target="_blank">http://www.worthington-bio</a>chem.com / cls / match.php) può essere usato per trovare batch disponibili con una composizione che ha dimostrato di essere adatto per l&#39;isolamento di miociti atriali umani. Attualmente usiamo collagenasi di tipo I con 250 U / mg attività collagenasi, 345 U / mg attività caseinase, 2.16 U / mg attività clostripain e 0.48 U / mg attività trittico (lotto # 49H11338). Le cellule ottenute utilizzando la procedura descritta in questo manoscritto possono essere utilizzati entro 8 ore per studi di patch-clamp, Ca 2 + transitori misurazioni e una combinazione di entrambi. 15 Inoltre, queste cellule consentono misure di contrazione cellulare in risposta alla stimolazione del campo elettrico o stimolazione elettrica usando la pipetta di patch-clamp (osservazioni non pubblicate).

Studiare le proprietà elettrofisiologiche dei canali ionici cardiaci con la tecnica del patch-clamp e l&#39;esplorazione di cellulare Ca 2 + di movimentazione anomalie richiedono cardiomiociti isolati. Questi sono solitamente ottenuti dopo l&#39;esposizione in vitro di campioni di tessuto cardiaco di enzimi digestivi (collagenasi, ialuronidasi, peptidasi ecc). Dal momento che il primo rapporto di isolamento di miociti cardiaci vitali nel 1955 2 una grande quantità di protocolli è stato sviluppato al fine di raccogliere singoli cardiomiociti atriali e ventricolari da specie diverse, tra cui topo, ratto, coniglio, cane, cavia e umani. In questa recensione ci concentriamo su isolamento dei miociti atriali umani. Per quanto riguarda le procedure di isolamento dei miociti da altre specie ci si riferisce al &quot;Worthington Tissue guida dissociazione&quot; fornito da Worthington Biochemical Corp., USA ( <a href="http://www.tissuedissociation.com" target="_blank">www.tissuedissociation.com</a> ). et al. 3 Qui forniamo una descrizione step-by-step di una tecnica, che è tratto da un metodo pubblicato in precedenza, al fine di ottenere miociti atriali adatte non solo per gli esperimenti patch clamp ma anche per simultanei Ca2 + intracellulare misurazioni. 4-11

<table border="1">
 <tbody>
 <tr>
 <td> &nbsp;</td>
 <td> &nbsp;</td>
 <td> &nbsp;</td>
 <td>Transport solution</td>
 </tr>
 <tr>
 <td>Albumin</td>
 <td>Sigma-Aldrich</td>
 <td>A3059</td>
 <td>Storage solution: 1%</td>
 </tr>
 <tr>
 <td>BDM</td>
 <td>Sigma-Aldrich</td>
 <td>31550</td>
 <td>Transport solution: 30</td>
 </tr>
 <tr>
 <td>DL-b-Hydroxy-butyric acid</td>
 <td>Sigma-Aldrich</td>
 <td>H6501</td>
 <td>Storage solution: 10</td>
 </tr>
 <tr>
 <td>Glucose</td>
 <td>Sigma-Aldrich</td>
 <td>G8270</td>
 <td>Transport solution: 20 
 Ca2+-free solution: 20 
 Enzyme solution E1 and E2: 20 
 Storage solution: 10</td>
 </tr>
 <tr>
 <td>L-Glutamic acid</td>
 <td>Sigma-Aldrich</td>
 <td>G1251</td>
 <td>Storage solution: 70</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>Merck</td>
 <td>1049360250</td>
 <td>Transport solution: 10 
 Ca2+-free solution: 10 
 Enzyme solution E1 and E2: 10 
 Storage solution: 20</td>
 </tr>
 <tr>
 <td>KH2PO4</td>
 <td>Sigma-Aldrich</td>
 <td>P5655</td>
 <td>Transport solution: 1.2 
 Ca2+-free solution: 1.2 
 Enzyme solution E1 and E2: 1.2 
 Storage solution: 10</td>
 </tr>
 <tr>
 <td>MgSO4</td>
 <td>Sigma-Aldrich</td>
 <td>M9397</td>
 <td>Transport solution: 5 
 Ca2+-free solution: 5 
 Enzyme solution E1 and E2: 5</td>
 </tr>
 <tr>
 <td>MOPS</td>
 <td>Sigma-Aldrich</td>
 <td>M1254</td>
 <td>Transport solution: 5 
 Ca2+-free solution: 5 
 Enzyme solution E1 and E2: 5</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Sigma-Aldrich</td>
 <td>S3014</td>
 <td>Transport solution: 100 
 Ca2+-free solution: 100 
 Enzyme solution E1 and E2: 100</td>
 </tr>
 <tr>
 <td>Taurin</td>
 <td>Sigma-Aldrich</td>
 <td>86330</td>
 <td>Transport solution: 50 
 Ca2+-free solution: 50 
 Enzyme solution E1 and E2: 50 
 Storage solution: 10</td>
 </tr>
 <tr>
 <td>Collagenase I</td>
 <td>Worthington</td>
 <td>4196</td>
 <td>Enzyme solution E1 and E2: 286 U/ml</td>
 </tr>
 <tr>
 <td>Protease XXIV</td>
 <td>Sigma-Aldrich</td>
 <td>P8038</td>
 <td>Enzyme solution E1 and E2: 5 U/ml*</td>
 </tr>
 <tr>
 <td>pH</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Transport solution: 7.00 
 Ca2+-free solution: 7.00 
 Enzyme solution E1 and E2: 7.00 
 Storage solution: 7.40</td>
 </tr>
 <tr>
 <td>adjusted with</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Transport solution: 1 M NaOH 
 Ca2+-free solution: 1 M NaOH 
 Enzyme solution E1 and E2: 1 M NaOH 
 Storage solution: 1 M KOH</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Concentrations in mM unless otherwise stated. BDM, 2,3-Butanedione monoxime. *Protease XXIV is included in Enzyme solution E1 only.</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table I. Solutions.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nylon mesh (200 &mu;m)</td>
 <td>VWR-Germany</td>
 <td>510-9527</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Jacketed reaction beaker</td>
 <td>VWR</td>
 <td>KT317000-0050</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table II. Specific equipment.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dimethyl-sulphoxide</td>
 <td>Sigma-Aldrich</td>
 <td>D2650</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fluo-3 AM (special packaging)</td>
 <td>Invitrogen</td>
 <td>F-1242</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Pluronic F-127</td>
 <td>Invitrogen</td>
 <td>P6867</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table III. Substances for loading of myocytes with Fluo-3 AM.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Bath solution</td>
 </tr>
 <tr>
 <td>4-aminopyridine*</td>
 <td>Sigma-Aldrich</td>
 <td>A78403</td>
 <td>Bath solution: 5</td>
 </tr>
 <tr>
 <td>BaCl2*</td>
 <td>Sigma-Aldrich</td>
 <td>342920</td>
 <td>Bath solution: 0.1</td>
 </tr>
 <tr>
 <td>CaCl2 &times; 2H2O</td>
 <td>Sigma-Aldrich</td>
 <td>C5080</td>
 <td>Bath solution: 2</td>
 </tr>
 <tr>
 <td>Glucose</td>
 <td>Sigma-Aldrich</td>
 <td>G8270</td>
 <td>Bath solution: 10</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma-Aldrich</td>
 <td>H9136</td>
 <td>Bath solution: 10</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>Merck</td>
 <td>1049360250</td>
 <td>Bath solution: 4</td>
 </tr>
 <tr>
 <td>MgCl &times; 6H2O</td>
 <td>Sigma-Aldrich</td>
 <td>M0250</td>
 <td>Bath solution: 1</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Sigma-Aldrich</td>
 <td>S3014</td>
 <td>Bath solution: 140</td>
 </tr>
 <tr>
 <td>Probenecid</td>
 <td>Sigma-Aldrich</td>
 <td>P8761</td>
 <td>Bath solution: 2</td>
 </tr>
 <tr>
 <td>pH</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Bath solution: 7.35</td>
 </tr>
 <tr>
 <td>adjusted with</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Bath solution: 1 M HCl</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table IV. Bath solution for patch-clamp. *4-aminopyridine and BaCl were included for voltage-clamp experiments only.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Pipette solution</td>
 </tr>
 <tr>
 <td>DL-aspartat K+-salt</td>
 <td>Sigma-Aldrich</td>
 <td>A2025</td>
 <td>Pipette solution: 92</td>
 </tr>
 <tr>
 <td>EGTA</td>
 <td>Sigma-Aldrich</td>
 <td>E4378</td>
 <td>Pipette solution: 0.02</td>
 </tr>
 <tr>
 <td>GTP-Tris</td>
 <td>Sigma-Aldrich</td>
 <td>G9002</td>
 <td>Pipette solution: 0.1</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma-Aldrich</td>
 <td>H9136</td>
 <td>Pipette solution: 10</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>Merck</td>
 <td>1049360250</td>
 <td>Pipette solution: 48</td>
 </tr>
 <tr>
 <td>MgATP</td>
 <td>Sigma-Aldrich</td>
 <td>A9187</td>
 <td>Pipette solution: 1</td>
 </tr>
 <tr>
 <td>Na2ATP</td>
 <td>Sigma-Aldrich</td>
 <td>A2383</td>
 <td>Pipette solution: 4</td>
 </tr>
 <tr>
 <td>Fluo-3**</td>
 <td>Invitrogen</td>
 <td>F3715</td>
 <td>Pipette solution: 0.1</td>
 </tr>
 <tr>
 <td>pH</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>7.20</td>
 </tr>
 <tr>
 <td>adjusted with</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>1 M KOH</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table V. Pipette solution for patch-clamp*. *On experimental days pipette solution is stored on ice until use. **Fluo-3 is added from a 1 mM stock solution on experimental days.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

isolation of human atrial myocytes for simultaneous measurements of ca2+ transients and membrane currents

Lo studio delle proprietà elettrofisiologiche dei canali ionici cardiaci con la tecnica del patch-clamp e l&#39;esplorazione delle cardiaco cellulare Ca 2 + movimentazione anomalie richiede cardiomiociti isolati. Inoltre, la possibilità di indagare miociti da pazienti che utilizzano queste tecniche è un requisito prezioso per delucidare le basi molecolari di malattie cardiache come la fibrillazione atriale (AF). 1 Qui si descrive un metodo per l&#39;isolamento di miociti atriali umani che sono adatti per entrambi studi di patch-clamp e misurazioni simultanee di concentrazioni intracellulari di Ca 2 +. In primo luogo, appendici atriali giuste ottenuti da pazienti sottoposti a chirurgia a cuore aperto sono tagliate in piccoli pezzi di tessuto (&quot;metodo chunk&quot;) e lavati a Ca 2 + soluzione priva. Poi i pezzi di tessuto vengono digeriti in collagenasi e proteasi contenente soluzioni con 20 mM Ca 2 +. Successivamente, i miociti isolati sono harvested per filtrazione e centrifugazione della sospensione tessuto. Infine, il Ca 2 + concentrazione nella soluzione di conservazione delle cellule viene modificata gradualmente a 0,2 mM. Discuteremo brevemente il significato di Ca 2 + e Ca 2 + tampone durante il processo di isolamento e anche fornire registrazioni rappresentativi di potenziali d&#39;azione e correnti di membrana, entrambi insieme con contestuale Ca 2 + misure di transienti, eseguita in questi miociti isolati.

Figura 2A mostra tre esempi rappresentativi di isolati miociti atriali diritto umano. Per quantificare il rendimento delle cellule abbiamo pipetta 10 ml di sospensione cellulare (passo 5.5) su un vetrino da microscopio CellFinder ( <a href="http://www.antenna.nl/microlab/index-uk.html" target="_blank">http://www.antenna.nl/microlab/index-uk.html</a> ). Produzioni di cellule mediata della figura 2B indicano chiaramente che vi è una tendenza a produzioni di cellule inferiori in FA cronica (CAF) campioni di pazienti (tubo A: 16.5 ± 3.1 cells/10 microlitri (n = 29) vs 5.1 ± 2.3 cells/10 microlitri (n = 10) in SR e Caf, rispettivamente, p &lt;0,05; tubo B: 17.9 ± 3.9 cells/10 ml (n = 29) vs 5.9 ± 2.0 cells/10 ml (n = 9) in SR e Caf, rispettivamente, p = 0,107). Esempi rappresentativi di azione-potenziali misurazioni e registrazioni simultanee di Ca 2 + citosolico transienti sono riportati nella figura 3. In circa il 90% delle cellule indagati, tha azione-potenziale-triggered Ca 2 + rilascio provoca chiare e regolari contrazioni delle cellule. Come riportato in precedenza, il potenziale di membrana a riposo, che è un indicatore accettato per l&#39;integrità delle cellule, in media circa -73,9 ± 2,7 mV (n = 23/10 miociti / pazienti) e -77,7 ± 1,8 mV (n = 19/8 miociti / pazienti ) in SR e Caf, rispettivamente (p&gt; 0,05). 15 La figura 4 mostra le registrazioni rappresentative simultanee di voltaggio-dipendenti di tipo L di Ca 2 + correnti e Ca 2 + citosolico transienti. Applicazione del non selettivo β-adrenergici isoprenalina agonista (1 mM) aumenta ampiezze di entrambi I Ca, L e Ca 2 + citosolico transitori, suggerendo intatta β-adrenergico cascata di trasduzione del segnale. <img alt="Figura 1" fo:content-width="4in" fo:src="/files/ftp_upload/50235/50235fig1highres.jpg" src="/files/ftp_upload/50235/50235fig1.jpg" /> Figura 1. Diagramma di flusso dei miociti Fluo-03:00 caricando protocollo (vedi passo 6,1-6,5). m / v, massa / volume. <img alt="Figura 2" fo:content-width="4in" fo:src="/files/ftp_upload/50235/50235fig2highres.jpg" src="/files/ftp_upload/50235/50235fig2.jpg" /> Figura 2. A, con separazione dei diritti umani miociti atriali dopo un&#39;ora in una soluzione di archiviazione. B, Media ± SEM della produzione di cellule contate in 10 ml di cellule in soluzione di conservazione (vedi punto 5.5). n si riferisce al numero di preparati di ciascun gruppo. * P &lt;0.05. <img alt="Figura 3" fo:content-width="4in" fo:src="/files/ftp_upload/50235/50235fig3highres.jpg" src="/files/ftp_upload/50235/50235fig3.jpg" /> Figura 3. Registrazioni rappresentative di azione-potenziale-triggered Ca 2 + transitori (CAT) in uno dei miociti atriali da un ritmo sinusale e un chronic paziente fibrillazione atriale. Top: corrente iniettata membrana (I M) utilizzato per la stimolazione (0,5 Hz). Sotto: Registrazione simultanea di potenziale di membrana (V M), e il gatto scatenato (in basso). (Rappresentato nuovamente con il permesso di Voigt et al. 2012) 15 <img alt="Figura 4" fo:content-width="4in" fo:src="/files/ftp_upload/50235/50235fig4highres.jpg" src="/files/ftp_upload/50235/50235fig4.jpg" /> Figura 4. Registrazioni rappresentativi del (1 mM) effetto isoprenalina su L-tipo Ca 2 + corrente attivata da Ca 2 + transitori (CAT) in uno dei miociti atriali da un ritmo sinusale e un paziente fibrillazione atriale cronica. Top: tensione-clamp protocollo (0,5 Hz). Sotto: Registrazione simultanea di membrana totale netta corrente (I M), riflettendo prevalentemente di tipo L Ca 2 Cat. + corrente (al centro) e attivato (in basso). (Rappresentato nuovamente con il permesso di Voigt,et al. 2012) 15 <img src="/files/ftp_upload/50235/50235table1.jpg" alt="Tabella 1" fo:content-width="4in" fo:src="/files/ftp_upload/50235/50235table1highres.jpg" /> Tabella I. Solutions. <img src="/files/ftp_upload/50235/50235table2.jpg" alt="Tabella 2" fo:content-width="2in" fo:src="/files/ftp_upload/50235/50235table2highres.jpg"/> Tabella II. Attrezzature specifiche. <img src="/files/ftp_upload/50235/50235table3.jpg" alt="Tabella 3" fo:content-width="2in" fo:src="/files/ftp_upload/50235/50235table3highres.jpg"/> Tabella III. Sostanze per il carico dei miociti con Fluo-3 AM. <img src="/files/ftp_upload/50235/50235table4.jpg" alt="Tabella 4" fo:content-width = &quot;2.5in&quot; fo: src = &quot;/ files/ftp_upload/50235/50235table4highres.jpg&quot; /&gt; Tabella IV. Soluzione del bagno di patch-clamp. <img src="/files/ftp_upload/50235/50235table5.jpg" alt="Tabella 5" fo:content-width="2.5in" fo:src="/files/ftp_upload/50235/50235table5highres.jpg"/> Tabella soluzione Pipetta V. per patch-clamp *.

Watch this Scientific Journal Video about Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents at JoVE.com

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

Descriviamo l&#39;isolamento di miociti atriali umani, che possono essere utilizzati per Ca intracellulare<sup&gt; 2 +</sup&gt; Misure in combinazione con studi elettrofisiologici di patch-clamp.

Isolamento dei miociti atriali umane per misure simultanee di Ca<sup&gt; 2 +</sup&gt; Transitori e correnti di membrana

The study of  electrophysiological properties of cardiac ion channels with the patch-clamp  technique and the exploration of cardiac cellular Ca2+ ...

isolation-human-atrial-myocytes-for-simultaneous-measurements-ca2

Research

JoVE Journal

Biology

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

20.2K Views.  University of Duisburg-Essen. Descriviamo l'isolamento di miociti atriali umani, che possono essere utilizzati per Ca intracellulare 2 + Misure in combinazione con studi elettrofisiologici di patch-clamp.

Video: Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

Cultura primario di miociti adulti Cuore Rat

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Ricerca

Biologia

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

L&#39;isolamento e la manipolazione genetica di adulti miociti cardiaci per confocale Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Tecniche di Imaging Ca 2 + Segnalazione di sperma umano

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

Isolamento e registrazioni dei canali Kv in cardiomiociti murini atriale e ventricolare

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

L&#39;isolamento di mitocondri da minime quantità di muscolo scheletrico del mouse per misurazioni High Throughput micropiastre respiratorie

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

Sviluppo e caratterizzazione di<em&gt; In Vitro</em&gt; Microvasi Rete e misurazioni quantitative di endoteliale [Ca<sup&gt; 2+</sup&gt;]<sub&gt; i</sub&gt; E ossido nitrico Produzione

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

Metodi per l&#39;isolamento, la cultura, e la caratterizzazione funzionale di seno-atriale Nodo miociti da topi adulti

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

Misurazione simultanea di mitocondriale calcio e mitocondriale potenziale di membrana in cellule vive di Microscopia fluorescente

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Simultaneous Mapping and Quantitation of Ribonucleotides in Human Mitochondrial DNA

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using short synthetic DNA fragments or plasmids as templates and then extrapolating the results to the whole genome. Alternatively, the number of ribonucleotides present in a genome may be estimated using alkaline gels or Southern blots. More recent in vivo approaches employ Next-generation sequencing allowing genome-wide mapping of ribonucleotides, providing the position and identity of embedded ribonucleotides. However, they do not allow quantitation of the number of ribonucleotides which are incorporated into a genome. Here we describe how to simultaneously map and quantitate the number of ribonucleotides which are incorporated into human mitochondrial DNA in vivo by Next-generation sequencing. We use highly intact DNA and introduce sequence specific double strand breaks by digesting it with an endonuclease, subsequently hydrolyzing incorporated ribonucleotides with alkali. The generated ends are ligated with adapters and these ends are sequenced on a Next-generation sequencing machine. The absolute number of ribonucleotides can be calculated as the number of reads outside the recognition site per average number of reads at the recognition site for the sequence specific endonuclease. This protocol may also be utilized to map and quantitate free nicks in DNA and allows adaption to map other DNA lesions that can be processed to 5&#180;-OH ends or 5&#180;-phosphate ends. Furthermore, this method can be applied to any organism, given that a suitable reference genome is available. This protocol therefore provides an important tool to study DNA replication, 5&#180;-end processing, DNA damage, and DNA repair.

Here we describe a method amenable to simultaneously quantitate and genome-wide map ribonucleotides in highly intact DNA at single-nucleotide resolution, combining enzymatic cleavage of genomic DNA with its alkaline hydrolysis and subsequent 5&#180;-end sequencing.

Mappatura simultanea e quantificazione di ribonucleotidi del DNA mitocondriale umano

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

Misurazioni simultanee di calcio intracellulare e potenziale di membrana nell'endotelio cerebrale Mouse appena isolato ed intatto

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...