Questo lavoro è stato sostenuto dall&#39;UE (STREP Progetto 241.577 &quot;GRANDE CUORE,&quot; 7 ° Programma quadro europeo, CP), Menarini internazionale Operations Luxembourg (AM), Telethon GGP07133 (CP) e Gilead Sciences (AM).

I protocolli sperimentali su tessuti umani sono stati approvati dal comitato etico di Careggi University-Hospital (2006/0024713; rinnoverà maggio 2009). Ogni paziente ha dato il consenso informato scritto. 1. Soluzioni agricole per la preparazione Le soluzioni sono descritte nella Tabella 1. Un diagramma di flusso semplificato della procedura di isolamento cellulare è situato nella Figura 1. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td colspan="2"> Soluzione </td><td> CP </td><td> DB </td><td> KB </td><td> TB </td><td> PS </td><td> EB1 </td><td> EB2 </td></tr><tr><td rowspan="23"> Reattivo (mm) </td><td> KH 2 PO 4 </td><td> 50 </td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> MgSO 4 </td><td> 8 </td><td> 1.2 </td><td> 5 </td><td></td><td></td><td> 1.2 </td><td> 1.2 </td></tr><tr><td> HEPES </td><td> 10 </td><td></td><td></td><td> 10 </td><td> 10 </td><td></td><td></td></tr><tr><td> adenosina </td><td> 5 </td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> glucosio </td><td> 140 </td><td> 10 </td><td> 20 </td><td> 10 </td><td></td><td> 10 </td><td> 10 </td></tr><tr><td> mannitolo </td><td> 100 </td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> taurina </td><td> 10 </td><td> 20 </td><td> 5 </td><td></td><td></td><td> 20 </td><td> 20 </td></tr><tr><td> NaCl </td><td></td><td> 113 </td><td></td><td> 136 </td><td></td><td> 113 </td><td> 113 </td></tr><tr><td> KCl </td><td></td><td> 4.7 </td><td> 85 </td><td> 5.4 </td><td> 25 </td><td> 4.7 </td><td> 4.7 </td></tr><tr><td> MgCl 2 </td><td></td><td> &lt;/ Td&gt; <td></td><td> 1.2 </td><td> 5 </td><td></td><td></td></tr><tr><td> KH 2 PO 4 </td><td></td><td> 0.6 </td><td> 30 </td><td></td><td></td><td> 0.6 </td><td> 0.6 </td></tr><tr><td> Na 2 HPO 4 </td><td></td><td> 0.6 </td><td></td><td></td><td></td><td> 0.6 </td><td> 0.6 </td></tr><tr><td> NaHCO 3 </td><td></td><td> 12 </td><td></td><td></td><td></td><td> 12 </td><td> 12 </td></tr><tr><td> KHCO 3 </td><td></td><td> 10 </td><td></td><td></td><td></td><td> 10 </td><td> 10 </td></tr><tr><td> Na-piruvato </td><td></td><td> 4 </td><td></td><td></td><td></td><td> 4 </td><td> 4 </td></tr><tr><td> BDM </td><td></td><td> 10 </td><td></td><td></td><td></td><td> 10 </td><td> 10 </td></tr><tr><td> BHBA </td><td></td><td></td><td> 5 &lt;/ Td&gt; <td></td><td></td><td></td><td></td></tr><tr><td> acido succinico </td><td></td><td></td><td> 5 </td><td></td><td></td><td></td><td></td></tr><tr><td> EGTA </td><td></td><td></td><td> 0.5 </td><td></td><td></td><td></td><td></td></tr><tr><td> K 2-ATP </td><td></td><td></td><td> 2 </td><td></td><td></td><td></td><td></td></tr><tr><td> acido piruvico </td><td></td><td></td><td> 5 </td><td></td><td></td><td></td><td></td></tr><tr><td> creatina </td><td></td><td></td><td> 5 </td><td></td><td></td><td></td><td></td></tr><tr><td> KMES </td><td></td><td></td><td></td><td></td><td> 115 </td><td></td><td></td></tr><tr><td rowspan="2"> Enzimi (U / ml) </td><td> Collagenase tipo V </td><td></td><td></td><td></td><td></td><td></td><td> 250 &lt;/ Td&gt; <td> 250 </td></tr><tr><td> Protease Tipo XXIV </td><td></td><td></td><td></td><td></td><td></td><td> 4 </td><td></td></tr><tr><td> pH </td><td></td><td> 7.4 KOH </td><td> 7.3 NaOH </td><td> 7.1 KOH </td><td> 7.35 NaOH </td><td> 7.2 KOH </td><td> 7.3 NaOH </td><td> 7.3 NaOH </td></tr></tbody></table> . Tabella 1 Soluzioni utilizzati per la raccolta dei campioni, l&#39;isolamento delle cellule e caratterizzazione funzionale di miociti CP = soluzione cardioplegica.; DB = buffer di dissociazione; KB = soluzione Kraft-Brühe; TB = tampone Tyrode; PS = soluzione di pipetta; EB1 = tampone enzimatico 1; EB2 = tampone enzima 2. <ol><li> Preparare la soluzione cardioplegica (CP). Soluzione CP può essere conservato a 4 ° C per 1 settimana. </li><li> Preparare Ca 2 + libero-tampone di dissociazione (DB). Questa soluzione deve essere utilizzata entro la giornata. </li><li> Preparare la soluzione Kraft-Brühe (KB). KB soluzione può essere conservato a 4 ° C per un massimo di 1 week. </li><li> Preparare Ca 2 buffer di Tyrode libero + (TB). Questa soluzione deve essere utilizzata entro la giornata. </li><li> Filtrare con usando filtri siringa prima dell&#39;uso. </li><li> Preparare il dispositivo di digestione (Figura 2), un contenitore raschiatura fatta di due spazzole affacciate di elastomero siliconico, una delle quali in rotazione da un motore elettrico. Il dispositivo di digestione è su misura. Dettagli sul dispositivo digestione sono in Figura 8; immagini del dispositivo sono nelle figure 2 C e 2D. Lavare la camera di tessuto con il 70% di etanolo e acqua. </li></ol> 2. Raccolta ed elaborazione dei Campioni Infarto <ol><li> Versare 40 ml di soluzione cardiplegic (CP) in un tubo da 50 ml e conservare in ghiaccio per trasporto campione dalla sala operatoria al laboratorio di isolamento cellulare. </li><li> Raccogliere il campione del miocardio ventricolare dalla sala operatoria subito dopo l&#39;asportazione, lavarlo con ghiaccio freddoSoluzione CP e memorizzarlo nel tubo. Utilizzare campioni endocardiche asportato dal setto ventricolare tra superiore durante l&#39;intervento chirurgico a cuore aperto, la ponderazione&gt; 100 mg. </li><li> Trasferire rapidamente il campione alla zona di laboratorio; avviare l&#39;elaborazione del campione entro 10 minuti da un campione escissione. </li><li> Pur mantenendo il campione di tampone a freddo ghiaccio CP, rimuovere delicatamente lo strato fibrotico endocardial con le forbici sottili allo stereomicroscopio; poi, tagliare il tessuto miocardico a piccoli pezzi (2-3 mm di lunghezza). A seconda delle dimensioni del campione di tessuto, tagliare un importo complessivo di miocardio ventricolare tra 100 mg e 1 g per ogni isolamento. </li><li> Al termine della macinazione tessuto, trasferire i pezzi del miocardio nel dispositivo di digestione, con ghiaccio pulito soluzione CP freddo. Evitare di riempire tutto il volume tra le due spazzole di silicio (3-4 ml) con pezzi del miocardio, utilizzando non più di 1 g di tessuto totale. </li></ol> 3. Lavaggio e Digestione del miocardio Chunks <ol><li> A poppaer i pezzi vengono trasferiti nella camera raschiando il dispositivo di digestione, modificare il buffer CP nella camera con il freddo Ca 2 + buffer di dissociazione-free (DB). </li><li> Posizionare il dispositivo di digestione in un bagno termostatico, in modo che la camera di essere in contatto con l&#39;acqua riscaldata nella vasca (Figura 1). Impostare la vasca di 37,5 ° C e accenderla, al fine di aumentare lentamente la temperatura della camera di tessuto. Accendere il motore del dispositivo di digestione, impostando la velocità di rotazione di 1 giro / secondo. </li><li> Eseguire 3 cicli di lavaggio con DB, cambiando la soluzione nella camera pulita con DB ogni 8 min. Il DB è riscaldata (37 ° C) e ossigeno saturo prima di entrare in contatto con i pezzi del miocardio. </li><li> Preparare tampone enzimatico 1 (EB1) con l&#39;aggiunta di 250 U / ml di collagenasi di tipo V e 4 U / ml Protease Tipo XXIV alla soluzione DB. Preparare tampone enzima 2 (EB2) con l&#39;aggiunta di 250 U / ml collagenasi di tipo V di soluzione DB. Warm up (37 ° C) e oxygenate EB1 ed EB2. </li><li> Eseguire due cicli di 12 min di digestione nel dispositivo digestione rotante con 100% EB1 ossigenato (a 37 ° C). Ad ogni ciclo, utilizzare ~ 3 ml di EB1. Rimuovere la soluzione pipetta aspirazione e scartarla dopo ogni ciclo. </li><li> Preparare 6 provette da 15 ml per la raccolta di cellule e ~ 80 ml di freddo (4 ° C) KB soluzione per eluire i buffer. </li><li> Effettuare un primo ciclo digestione 15 min con 3 ml 100% EB2 ossigenata a 37 ° C. Dopo il ciclo di digestione, raccogliere la soluzione contenente i primi miociti dissociate in una provetta da 15 ml e diluire la sospensione cellulare con 12 ml di soluzione fredda KB. Conservare il piatto tubo a temperatura ambiente. </li><li> Diluire la soluzione EB2 rimanente con una quantità uguale di DB per dimezzare la concentrazione di collagenasi V per i seguenti cicli di digestione. </li><li> Eseguire altri cicli 5 12 min digestione con 3 EB2 ml a 37 ° C; dopo ciascuno di loro raccolta di miociti contenente tampone in un tubo da 15 ml ediluire con 12 ml di soluzione KB. Conservare le provette cella contenente 6 a temperatura ambiente per 30 min. </li></ol> 4. Resuspension cellulare e Ca 2 + riadattamento <ol><li> Aggiungere 1 mg / ml di albumina di siero bovino (BSA) per 20 ml tampone Ca 2 +-Tyrode libero (TB). Filtrare la soluzione. </li><li> Centrifugare le sei tubi conici miociti contenente 100 xg per 5 minuti per forzare miociti per risolvere. Rimuovere il surnatante e risospendere le cellule in ciascun tubo con un ammontare variabile (1-3 ml, a seconda della resa) di BSA contenente TB a RT. </li><li> Aumentare gradualmente Ca 2 + concentrazione nella memoria di cella contenente aggiungendo piccole aliquote di 100 mmol / L CaCl 2 soluzione. Nella prima e nella seconda fase di Ca 2 + concentrazione viene aumentata fino a 50 mmol / L e 100 mmol / L, rispettivamente. I seguenti passaggi Ca 2 + addizione vengono eseguiti ogni 5 minuti e la concentrazione viene sollevato da 100 micromol / L ad ogni passo toa concentrazione finale di 0,9 mmol / L. </li><li> Valutare la resa della procedura di isolamento. Trasferire 0,5 ml di soluzione contenente miociti sulla camera inferiore di vetro di un microscopio. Valutare 15 campi di microscopio a 10x obiettivo e calcolare la percentuale di miociti sani (ad esempio cellule a forma di bastoncello, con striature chiare e senza inclusioni significative, Figura 2). Il rendimento atteso è di circa il 20%. </li></ol> 5. Valutazione funzionale dei cardiomiociti isolati. Il protocollo che segue è un esempio di valutazione funzionale dei cardiomiociti umani tra cui registrazioni simultanee di potenziali d&#39;azione e intracellulare Ca 2 + flussi. <ol><li> Preparare la soluzione di pipetta (PS) per esperimenti di patch clamp in configurazione di patch perforato. La soluzione può essere conservata a -20 ° C per 3 mesi. </li><li> Aggiungere 1,8 mmol / L CaCl 2 a Ca 2 + tampone Tyrode-free (TB). Use questa soluzione per superfusione dei cardiomiociti durante esperimenti di patch clamp / fluorescenza. </li><li> Trasferire 1 ml di sospensione cellulare in una provetta da 1,5 ml e aggiungere 10 micromol / L Fluoforte e 10 microlitri Powerload Concentrato. Incubare per 30 minuti a RT. Successivamente, impostare il tubo in posizione verticale e lasciare la cella accontentarsi di 5 min; risospendere le cellule in Ca 2 + contenente TB. </li><li> Trasferire 0,25 ml di sospensione cellulare in un piccolo (0,5 ml), microscopio temperatura controllata montato camera di registrazione, superfuse per gravità con un sistema microperfusor riscaldato a una velocità di flusso di 0,3 ml / min (temperatura: 37 ± 0,5 ° C). </li><li> Con un estrattore micropipetta, preparare pipette di patch clamp con un diametro di punta di 3 a 5 micron e una resistenza da 3 a 4,5 m quando riempita con PS. </li><li> Aggiungere amfotericina B a un lotto di PS (250 mcg / ml) e usarlo per riempire gli elettrodi. </li><li> Selezionare una cella canna a forma di striature chiare, privo di inclusioni, form il sigillo giga e aspettare 5-10 minuti, fino a quando la resistenza accesso scende al di sotto di 20 mW. </li><li> Suscitare potenziali d&#39;azione in modalità pinza corrente utilizzando impulsi brevi (&lt;3 msec) a diverse frequenze di stimolazione (0,2 Hz, 0.5 Hz e 1 Hz, 1 min ciascuna frequenza). Durante la fase di registrazione, accendere l&#39;illuminazione in campo chiaro a 492 ± 3 nm e rilevare Fluoforte fluorescenza a 505-520 nm. Acquisire fluorescenza e membrana segnali di potenziali utilizzando Digidata 1440A e software pClamp 10.0. Ripetere la sequenza di registrazione più volte, se necessario; tuttavia, mantenere il tempo di registrazione totale inferiore a 15 minuti per ogni cella. </li></ol>

<ol>
	<li>Dow, J. W., Harding, N. G., Powell, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+cardiac+myocytes.+I.+Preparation+of+adult+myocytes+and+their+homology+with+the+intact+tissue.">Isolated cardiac myocytes. I. Preparation of adult myocytes and their homology with the intact tissue.</a> Cardiovascular Research. 15, 483-514 (1981).</li><li>Dow, J. W., Harding, N. G., Powell, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+cardiac+myocytes.+II.+Functional+aspects+of+mature+cells.">Isolated cardiac myocytes. II. Functional aspects of mature cells.</a> Cardiovascular Research. 15, 549-579 (1981).</li><li>Harding, S. 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=Species+dependence+of+contraction+velocity+in+single+isolated+cardiac+myocytes.">Species dependence of contraction velocity in single isolated cardiac myocytes.</a> Cardioscience. 1, 49-53 (1990).</li><li>Bustamante, J. O., Watanabe, T., Murphy, D. A., McDonald, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+single+atrial+and+ventricular+cells+from+the+human+heart.">Isolation of single atrial and ventricular cells from the human heart.</a> Canadian Medical Association Journal. 126, 791-793 (1982).</li><li>Beuckelmann, D. J., Nabauer, M., Erdmann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+calcium-current+in+isolated+human+ventricular+myocytes+from+patients+with+terminal+heart+failure.">Characteristics of calcium-current in isolated human ventricular myocytes from patients with terminal heart failure.</a> Journal of Molecular and Cellular Cardiology. 23, 929-937 (1991).</li><li>Beuckelmann, D. J., Nabauer, M., Erdmann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+calcium+handling+in+isolated+ventricular+myocytes+from+patients+with+terminal+heart+failure.">Intracellular calcium handling in isolated ventricular myocytes from patients with terminal heart failure.</a> Circulation. 85, 1046-1055 (1992).</li><li>Cohen, N. M., Lederer, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+current+in+single+human+cardiac+myocytes.">Calcium current in single human cardiac myocytes.</a> Journal of Cardiovascular Electrophysiology. 4, 422-437 (1993).</li><li>Beuckelmann, D. J., Nabauer, M., Erdmann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+of+K++currents+in+isolated+human+ventricular+myocytes+from+patients+with+terminal+heart+failure.">Alterations of K+ currents in isolated human ventricular myocytes from patients with terminal heart failure.</a> Circulation Research. 73, 379-385 (1993).</li><li>Virag, 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=The+slow+component+of+the+delayed+rectifier+potassium+current+in+undiseased+human+ventricular+myocytes.">The slow component of the delayed rectifier potassium current in undiseased human ventricular myocytes.</a> Cardiovascular Research. 49, 790-797 (2001).</li><li>Nanasi, P. P., Varro, A., Lathrop, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+human+ventricular+and+atrial+cardiomyocytes:+technical+note.">Isolation of human ventricular and atrial cardiomyocytes: technical note.</a> Cardioscience. 4, 111-116 (1993).</li><li>Benitah, J. P., 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=Slow+inward+current+in+single+cells+isolated+from+adult+human+ventricles.+Pflugers+Archiv.">Slow inward current in single cells isolated from adult human ventricles. Pflugers Archiv.</a> European Journal of Physiology. 421, 176-187 (1992).</li><li>Verkerk, A. O., 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=Ionic+mechanism+of+delayed+afterdepolarizations+in+ventricular+cells+isolated+from+human+end-stage+failing+hearts.">Ionic mechanism of delayed afterdepolarizations in ventricular cells isolated from human end-stage failing hearts.</a> Circulation. 104, 2728-2733 (2001).</li><li>Cerbai, 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=Characterization+of+the+hyperpolarization-activated+current,+I(f),+in+ventricular+myocytes+from+human+failing+heart.">Characterization of the hyperpolarization-activated current, I(f), in ventricular myocytes from human failing heart.</a> Circulation. 95, 568-571 (1997).</li><li>Kohncke, C., 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=Isolation+and+kv+channel+recordings+in+murine+atrial+and+ventricular+cardiomyocytes.">Isolation and kv channel recordings in murine atrial and ventricular cardiomyocytes.</a> Journal of Visualized Experiments: JoVE. , (2013).</li><li>Tomaselli, G. F., Marban, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+remodeling+in+hypertrophy+and+heart+failure.">Electrophysiological remodeling in hypertrophy and heart failure.</a> Cardiovascular Research. 42, 270-283 (1999).</li><li>Maron, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypertrophic+cardiomyopathy:+a+systematic+review.">Hypertrophic cardiomyopathy: a systematic review.</a> JAMA: The Journal of the American Medical Association. 287, 1308-1320 (2002).</li><li>Olivotto, I., 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=The+many+faces+of+hypertrophic+cardiomyopathy:+from+developmental+biology+to+clinical+practice.">The many faces of hypertrophic cardiomyopathy: from developmental biology to clinical practice.</a> Journal of Cardiovascular Translational Research. 2, 349-367 (2009).</li><li>Maron, M. S., 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=Hypertrophic+cardiomyopathy+is+predominantly+a+disease+of+left+ventricular+outflow+tract+obstruction.">Hypertrophic cardiomyopathy is predominantly a disease of left ventricular outflow tract obstruction.</a> Circulation. 114, 2232-2239 (2006).</li><li>Peeters, G. A., 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=Method+for+isolation+of+human+ventricular+myocytes+from+single+endocardial+and+epicardial+biopsies.">Method for isolation of human ventricular myocytes from single endocardial and epicardial biopsies.</a> The American Journal of Physiology. 268, 1757-1764 (1995).</li><li>Lippiat, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-cell+recording+using+the+perforated+patch+clamp+technique.">Whole-cell recording using the perforated patch clamp technique.</a> Methods Mol Biol. 491, 141-149 (2008).</li><li>Coppini, R., 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=Late+sodium+current+inhibition+reverses+electromechanical+dysfunction+in+human+hypertrophic+cardiomyopathy.">Late sodium current inhibition reverses electromechanical dysfunction in human hypertrophic cardiomyopathy.</a> Circulation. 127, 575-584 (2013).</li><li>Kuusisto, J., 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=Low-grade+inflammation+and+the+phenotypic+expression+of+myocardial+fibrosis+in+hypertrophic+cardiomyopathy.">Low-grade inflammation and the phenotypic expression of myocardial fibrosis in hypertrophic cardiomyopathy.</a> Heart. 98, 1007-1013 (2012).</li><li>Yan, G. X., 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=Phase+2+early+afterdepolarization+as+a+trigger+of+polymorphic+ventricular+tachycardia+in+acquired+long-QT+syndrome+:+direct+evidence+from+intracellular+recordings+in+the+intact+left+ventricular+wall.">Phase 2 early afterdepolarization as a trigger of polymorphic ventricular tachycardia in acquired long-QT syndrome : direct evidence from intracellular recordings in the intact left ventricular wall.</a> Circulation. 103, 2851-2856 (2001).</li><li>Yue, L., Feng, J., Li, G. R., Nattel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+outward+and+delayed+rectifier+currents+in+canine+atrium:+properties+and+role+of+isolation+methods.">Transient outward and delayed rectifier currents in canine atrium: properties and role of isolation methods.</a> The American Journal of Physiology. 270, 2157-2168 (1996).</li><li>Li, G. R., Feng, J., Yue, L., Carrier, M., Nattel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+two+components+of+delayed+rectifier+K++current+in+human+ventricular+myocytes.">Evidence for two components of delayed rectifier K+ current in human ventricular myocytes.</a> Circulation research. 78, 689-696 (1996).</li><li>Viswanathan, P. C., Shaw, R. M., Rudy, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+IKr+and+IKs+heterogeneity+on+action+potential+duration+and+its+rate+dependence:+a+simulation+study.">Effects of IKr and IKs heterogeneity on action potential duration and its rate dependence: a simulation study.</a> Circulation. 99, 2466-2474 (1999).</li><li>Volders, P. G., 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=Probing+the+contribution+of+IKs+to+canine+ventricular+repolarization:+key+role+for+beta-adrenergic+receptor+stimulation.">Probing the contribution of IKs to canine ventricular repolarization: key role for beta-adrenergic receptor stimulation.</a> Circulation. 107, 2753-2760 (2003).</li><li>Sanguinetti, M. C., Jurkiewicz, N. K., Scott, A., Siegl, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoproterenol+antagonizes+prolongation+of+refractory+period+by+the+class+III+antiarrhythmic+agent+E-4031+in+guinea+pig+myocytes.+Mechanism+of+action.">Isoproterenol antagonizes prolongation of refractory period by the class III antiarrhythmic agent E-4031 in guinea pig myocytes. Mechanism of action.</a> Circulation Research. 68, 77-84 (1991).</li><li>Coppini, R., 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=A+translational+approach+to+treatment+of+hypertrophic+cardiomyopathy:+pre-clinical+rationale+and+design+of+a+prospective+randomized+pilot+trial+with+ranolazine.">A translational approach to treatment of hypertrophic cardiomyopathy: pre-clinical rationale and design of a prospective randomized pilot trial with ranolazine.</a> Circulation. 125, 1 (2012).</li></ol>

Gli autori dichiarano di non avere interessi finanziari in competizione.

Abbiamo descritto e validato un metodo per isolare miociti vitali da campioni chirurgici di miocardio ventricolare umano. Partendo da protocolli precedentemente descritti che erano stati utilizzati con successo per cellule isolate da campioni chirurgici atriali, la tecnica per consentire la separazione dei miociti vitali singoli da malato miocardio ventricolare è stato sviluppato e perfezionato. I primi rapporti hanno mostrato che l&#39;isolamento dei singoli cardiomiociti da pezzi di atriale e ventricolare tessuto sel...

La dissezione delle proprietà elettrofisiologiche del miocardio è progredita notevolmente dopo lo sviluppo di tecniche per l&#39;isolamento singolo dei miociti cardiaci. Recenti progressi nella comprensione dei cardiaco eccitazione contrazione Coupling (CE-Coupling), sono stati resi possibili dalla capacità di isolare vitali singoli cardiomiociti che conservano tutte le proprietà fisiologiche del tessuto intatto. Metodi di patch clamp sono abitualmente impiegati per studiare la funzione e la modulazione farmacologica di correnti ioniche sarcolemma cardiaco. Registrazioni di dinamica del calcio intracellulare con Ca 2 + coloranti sensibili sono regolarmente eseguite su miociti cardiaci singoli da una varietà di modelli sani e malati, fornendo dati vitali sulla fisiologia del CE-Coupling nonché sulle alterazioni patologiche intracellulare Ca 2 + omeostasi che porta a danni meccanici ed aumento degli oneri aritmogena nelle malattie cardiache. Informazione da questi studi è fondamentale per la comprensione degli effetti elettrofisiologici e meccanici di farmaci in ambito clinico. Tuttavia, ci sono specie specifiche differenze nelle correnti transmembrana e nelle proteine ​​CE-coupling che rappresentano caratteristiche specifiche di potenziale cardiaco e meccanica cardiaca. Così, mentre gli studi di miociti isolati da mammiferi non umani hanno chiarito le proprietà biofisiche e ruoli fisiologici di specifici canali ionici transmembrana e proteine ​​EC-Coupling, non necessariamente forniscono modelli provvisti di miociti cardiaci umani. Isolamento dei miociti vitali di miocardio umano è quindi essenziale per comprendere appieno la fisiopatologia delle malattie cardiache e validare nuovi approcci terapeutici. Tessuto atriale umano è facilmente disponibile come appendici atriali sono spesso scartati durante le procedure chirurgiche. Studi quantitativi iniziali di adulti potenziali d&#39;azione cardiaci umani e cu ionicarrents impiegate cellule atriali enzimatica isolate 1-4. Registrazioni di potenziali d&#39;azione o correnti di isolate cellule ventricolari umane adulte sono stati successivamente segnalati 3,5-10. La maggior parte di questi studi hanno utilizzato cellule ottenute da cuori espiantati e utilizzati sia perfusione con collagenasi di un segmento di arteria coronaria o l&#39;esposizione di una rilevante quantità di tessuto asportato alla collagenasi per ottenere cellule isolate. Questi studi hanno permesso una caratterizzazione dettagliata di un numero di correnti ioniche transmembrana di cardiomiociti ventricolari umani da cuori sani e da pazienti con insufficienza cardiaca terminale. Registrazioni di tipo L Ca 2 + corrente (I Ca-L) 5-7, corrente di potassio verso l&#39;esterno transitoria (I) 8, corrente entrante potassio raddrizzatore (I κ1) 8, le varie componenti della corrente di potassio delayed rectifier (I κ ) 9 sono stati segnalati. Anticipi e raffinazione dila procedura di isolamento 10, ha consentito una chiara caratterizzazione della base ionica del potenziale aumento aritmogenico nello scompenso cardiaco terminale, che comprende l&#39;azione potenziale prolungamento 11, ritardato dopo depolarizzazioni 12 e aumentato divertente attuali 13 che porta alla depolarizzazione diastolica e battiti prematuri. Miociti cardiaci adulti sono normalmente isolati da piccoli animali da perfusione retrograda di tutto cuore con varie miscele enzimatiche, una tecnica che produce rendimenti elevati di Ca 2 + cellule-tolleranti 14. Isolamento di miociti cardiaci da frammenti di tessuto è intrinsecamente meno successo probabilmente a causa del limitato accesso di enzimi per singoli miociti rispetto a quella ottenuta mediante perfusione delle arterie coronarie. A causa della limitata disponibilità di cuori erogatori non utilizzati, l&#39;unico modo pratico per ottenere cellule ventricolari umane normali su base regolare è da digestio enzimatican dei piccolissimi frammenti di tessuto spesso asportati durante le procedure chirurgiche elettive. L&#39;unico modello di malattia umana che è stato ampiamente caratterizzato a livello cellulare è insufficienza cardiaca terminale, a causa della accessibilità ai cuori trapiantati. Tuttavia, insufficienza cardiaca terminale si verifica in una minoranza di pazienti e spesso comporta un percorso comune di grave rimodellamento delle cellule del miocardio, che è relativamente indipendente dalla causa sottostante 15. La capacità di valutare la funzione di singoli cardiomiociti di pazienti in una fase precedente non avendo della malattia è fondamentale per capire la fisiopatologia specifica delle diverse condizioni ereditarie o acquisite. La cardiomiopatia ipertrofica (HCM) è un esempio eloquente. HCM è un comune (1/500 persone) condizione cardiaca ereditaria caratterizzata da ipertrofia cardiaca, aumento alterazioni rischio e contrattili aritmogenici a causa di ostruzione del tratto di efflusso e disfunzione diastolica 16. Cardiomiociti da cuori HCM undergo un complesso di processi di rimodellamento che comportano cambiamenti nella struttura delle cellule (ipertrofia, allo sbando miofibrillare) e EC-Coupling 17. Tuttavia, la maggior informazioni di miociti erettile in HCM è venuto da modelli animali transgenici. Dal momento che solo una minoranza di pazienti HCM evolve verso l&#39;insufficienza cardiaca terminale e necessita di trapianto cardiaco, il cuore HCM sono molto raramente disponibili per l&#39;isolamento delle cellule con metodi standard. Tuttavia, almeno il 30% dei pazienti HCM sviluppano sintomi ostruttivi dovuti alla enorme flusso di sangue tratto ipertrofia settale alterazione deflusso durante la sistole (HCM) 18. Il più efficace opzione terapeutica disponibile per il sollievo di ostruzione in HCM è chirurgica del setto miectomia: durante questa procedura chirurgica, una porzione di dimensioni variabile del setto superiore viene rimosso approccio aortica trans. Questa porzione di setto ipertrofico è quindi disponibile per l&#39;isolamento delle cellule dal tessuto fresco. Un metodo per l&#39;isolamento di ventricula umanar miociti dai singoli, piccoli transvenous campioni di biopsia endomiocardica è stato precedentemente sviluppato e pubblicato 19. Abbiamo implementato un metodo per isolare singoli miociti del setto da campioni di miocardio ventricolare da pazienti sottoposti a chirurgia cardiaca, inclusi i pazienti con HCM sottoposti miectomia settale e pazienti sottoposti a procedure di sostituzione della valvola. Oltre a una descrizione dettagliata del protocollo di isolamento, elettrofisiologico rappresentante e Ca 2 + fluorescenza misurazioni sono presentati, dimostrando la vitalità dei miociti ventricolari isolati umani e la fattibilità di patch clamp e Ca2 + intracellulare studi.

<table border="0" cellpadding="0" cellspacing="0" width="1059">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Potassium phosphate monobasic (KH2PO4)</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:139px;"><a href="http://www.sigmaaldrich.com/catalog/product/sigma/p9791?lang=it&amp;region=IT">P9791&#160;</a></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium sulfate heptahydrate(MgSO4 * 7H2O)</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/m1880?lang=it&amp;region=IT">M1880&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/h3375?lang=it&amp;region=IT">H3375&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adenosine</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a9251?lang=it&amp;region=IT">A9251&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-(+)-Glucose</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/g8270?lang=it&amp;region=IT">G8270&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mannitol</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/m4125?lang=it&amp;region=IT">M4125&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taurine</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td>T0625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium hydroxide (KOH)</td>
			<td>Sigma-Aldrich</td>
			<td>P5958</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/s7653?lang=it&amp;region=IT">S7653</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/p9333?lang=it&amp;region=IT">P9333&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate dibasic (Na2HPO4)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/s7907?lang=it&amp;region=IT">S7907&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium bicarbonate (NaHCO3)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/s6297?lang=it&amp;region=IT">S6297&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium bicarbonate (KHCO3)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/237205?lang=it&amp;region=IT">237205</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/p2256?lang=it&amp;region=IT">P2256&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2,3-Butanedione monoxime</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/b0753?lang=it&amp;region=IT">B0753&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium hydroxide(NaOH)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/s8045?lang=it&amp;region=IT">S8045&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamic acid monopotassium salt monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/fluka/49601?lang=it&amp;region=IT">49601</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pyruvic acid</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/aldrich/107360?lang=it&amp;region=IT">107360</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-Hydroxybutyric acid</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/aldrich/166898?lang=it&amp;region=IT">166898</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adenosine 5&#8242;-triphosphate dipotassium salt dihydrate (K2-ATP)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a8937?lang=it&amp;region=IT">A8937</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Creatine</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/c0780?lang=it&amp;region=IT">C0780&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Succinic Acid</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/s3674?lang=it&amp;region=IT">S3674&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid (EGTA)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/e0396?lang=it&amp;region=IT">E0396&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumin from bovine serum</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a0281?lang=it&amp;region=IT">A0281</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium chloride (MgCl2)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/m8266?lang=it&amp;region=IT">M8266&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase from Clostridium histolyticum, Type V</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/c9263?lang=it&amp;region=IT">C9263&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase, Bacterial, Type XXIV</td>
			<td>Sigma-Aldrich</td>
			<td>P8038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium chloride solution, ~1 M in H2O</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/21115?lang=it&amp;region=IT">21115</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium chloride 0.1 M solution</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/53704?lang=it&amp;region=IT">53704</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium methanesulfonate</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/aldrich/83000?lang=it&amp;region=IT">83000</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FluoForte Reagent</td>
			<td>Enzo Life Sciences</td>
			<td>ENZ-52015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Powerload concentrate, 100X</td>
			<td>Life Technologies</td>
			<td>P10020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perfusion Fast-Step System</td>
			<td>Warner Instruments</td>
			<td>VC-77SP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amphotericin B solubilized</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a9528?lang=it&amp;region=IT">A9528&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multiclamp 700B patch-clamp amplifier</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digidata 1440A</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pClamp10.0&#160;</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="108">
			<td height="108" style="height:108px;">Digestion Device</td>
			<td>CUSTOM</td>
			<td>CUSTOM</td>
			<td style="width:255px;">The device is custome made in our laboratory using plastic tubes, cast Sylgard and a motor; it is described in detail in Fig 1 C-D and in Fig.7. We can provide further details if requested</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicone elastomer for the digestion device&#39;s brushes</td>
			<td>Dow Corning</td>
			<td>SYLGARD&#174; 184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Variable speed rotating motor for the digestion device</td>
			<td>Crouzet</td>
			<td>Crouzet 178-4765&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Mold for brushes casting</td>
			<td>N.A.</td>
			<td>N.A.</td>
			<td style="width:255px;">The mold is custom made from standard PTFE 2.5 cm diameter rods</td>
		</tr>
	</tbody>
</table>

isolation and functional characterization of human ventricular cardiomyocytes from fresh surgical samples

Cardiomiociti da cuori malati sono sottoposti a processi di rimodellamento complessi che coinvolgono cambiamenti nella struttura cellulare, accoppiamento eccitazione contrazione e correnti ioniche di membrana. Tali modifiche sono suscettibili di essere responsabile per l&#39;aumento del rischio aritmogena e le alterazioni contrattili che portano alla disfunzione sistolica e diastolica nei pazienti cardiopatici. Tuttavia, la maggior informazioni sulle alterazioni della funzione miociti nelle malattie cardiache proviene da modelli animali. Qui descriviamo e validare un protocollo per isolare miociti vitali da piccoli campioni chirurgici di miocardio ventricolare da pazienti sottoposti a interventi di chirurgia cardiaca. Il protocollo è descritto in dettaglio. Misure elettrofisiologiche e calcio intracellulare sono segnalati per dimostrare la fattibilità di un numero di misurazioni singola cellula in cardiomiociti ventricolari umane ottenute con questo metodo. Il protocollo ha riferito chere possono essere utili per le future ricerche di base cellulare e molecolare delle alterazioni funzionali del cuore umano in presenza di differenti malattie cardiache. Inoltre, questo metodo può essere utilizzato per identificare nuovi bersagli terapeutici a livello cellulare e per testare l&#39;efficacia di nuovi composti in cardiomiociti umani, con valore traslazionale diretta.

Il metodo sopra descritto è stato impiegato per caratterizzare le alterazioni funzionali di cardiomiociti isolati dal setto interventricolare di pazienti con cardiomiopatia ipertrofica (HCM) che hanno subito un&#39;operazione miectomia, rispetto ai pazienti non in mancanza non ipertrofiche chirurgici 21. I risultati contenuti in questa sezione provengono da quel lavoro 21 e sono mostrati qui come un esempio di come questa tecnica può essere utilizzata per caratterizzare le alterazioni della funzi...

Watch this Scientific Journal Video about Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples at JoVE.com

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Le attuali conoscenze sulla base cellulare di malattie cardiache si basa principalmente su studi su modelli animali. Qui descriviamo e validare un nuovo metodo per ottenere singoli cardiomiociti vitali da piccoli campioni chirurgici di miocardio ventricolare umana. Miociti ventricolari umani possono essere utilizzati per studi elettrofisiologici e test della droga.

Isolamento e caratterizzazione funzionale di Human ventricolare Cardiomyocytes da campioni freschi chirurgici

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

isolation-functional-characterization-human-ventricular

Research

JoVE Journal

Medicine

17.1K Views.  University of Florence.  Le attuali conoscenze sulla base cellulare di malattie cardiache si basa principalmente su studi su modelli animali. Qui descriviamo e validare un nuovo metodo per ottenere singoli cardiomiociti vitali da piccoli campioni chirurgici di miocardio ventricolare umana. Miociti ventricolari umani possono essere utilizzati per studi elettrofisiologici e test della droga. 

Video: Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Paziente coltura cellulare derivato e isolamento di CD133<sup&gt; +</sup&gt; Putativi staminali alle cellule tumorali di melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Ricerca

Medicina

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

Isolamento di cancro cellule staminali da campioni umani di cancro alla prostata

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

Raccolta, isolamento e analisi citofluorimetrica di campioni endocervicali umano

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

Chronic Thromboembolic Pulmonary Hypertension and Assessment of Right Ventricular Function in the Piglet

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. Pulmonary Hypertension (PH) was induced in 3-week-old piglets by a progressive obstruction of the pulmonary vascular bed. A ligation of the left Pulmonary Artery (PA) was performed first through a mini-thoracotomy. Second, weekly embolizations of the right lower pulmonary lobe were done under fluoroscopic guidance with n-butyl-2-cyanoacrylate during 5 weeks. Mean Pulmonary Arterial Pressure (mPAP) measured by ritght heart catheterism, increased progressively, as well as Right Atrial pressure and Pulmonary Vascular Resistances (PVR) after 5 weeks compared to sham animals. Right Ventricular (RV) structural and functional remodeling were assessed by transthoracic echocardiography (RV diameters, RV wall thickness, RV systolic function). RV elastance and RV-pulmonary coupling were assessed by Pressure-Volume Loops (PVL) analysis with conductance method. Histologic study of the lung and the right ventricle were also performed. Molecular analyses on RV fresh tissues could be performed through repeated transcutaneous endomyocardial biopsies. Pulmonary microvascular disease in obstructed and unobstructed territories was studied from lung biopsies using molecular analyses and pathology. Furthermore, the reliability and the reproducibility was associated with a range of PH severity in animals. Most aspects of the human CTEPH disease were reproduced in this model, which allows new perspectives for the understanding of the underlying mechanisms (mitochondria, inflammation) and new therapeutic approaches (targeted, cellular or gene therapies) of the overloaded right ventricle but also pulmonary microvascular disease.

Chronic Thromboembolic Pulmonary Hypertension (CTEPH) and Right Ventricular (RV) dysfunction were induced in piglets by progressive obstruction of the pulmonary arteries. Consequences were remarkably similar to those observed in CTEPH patients. This animal model would be a very useful tool for pathophysiology and therapeutic experiments on CTEPH and RV failure.

Tromboembolica cronica Ipertensione polmonare e valutazione dei ventricolare destra Funzione nel Maialino

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. ...

Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum (SR) serves as a Ca2+ reservoir for contraction, which reuptakes intracellular Ca2+ during relaxation. The SR Ca2+ reserve available for beats is determinate for cardiac contractibility, and the removal of intracellular Ca2+ is critical for cardiac diastolic function. Under some pathophysiological conditions, such as diabetes and heart failure, impaired calcium clearance and SR Ca2+ store in cardiomyocytes may be involved in the progress of cardiac dysfunction. Here, we describe a protocol to evaluate SRCa2+ reserve and diastolic Ca2+ removal. Briefly, a single cardiomyocyte was enzymatically isolated, and the intracellular Ca2+ fluorescence indicated by Fura-2 was recorded by a calcium imaging system. To employ caffeine for inducing total SR Ca2+ release, we preset an automatic perfusion switch program by interlinking the stimulation system and the perfusion system. Then, the mono-exponential curve fitting was used for analyzing decay time constants of calcium transients and caffeine-induced calcium pulses. Accordingly, the contribution of the SR Ca2+-ATPase (SERCA) and Na+-Ca2+ exchanger (NCX) to diastolic calcium removal was evaluated.

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Here, we describe a protocol to evaluate sarcoplasmic reticulum Ca2+ reserve and diastolic calcium removal function in cardiomyocytes by a calcium imaging system.

Valutazione del reticolo sarcoplasmico calcio intracellulare e riserva diastolica calcio rimozione nei cardiomiociti ventricolari isolati

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum ...

Isolation and Cultivation of Adult Rat Cardiomyocytes

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise investigation of the behavior of these cells under specific treatments and environments is possible. This manuscript presents a protocol for successful isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC).
The rat is sacrificed by cervical dislocation under deep anesthesia. Then, the heart is extracted and the aorta is uncovered. Subsequently, perfusion on the Langendorff perfusion system with calcium depletion and collagenase treatment is performed. Afterwards, ventricular tissue gets minced, re-circulated, and filtered, followed by three centrifugation steps with gradual addition of CaCl2 until physiological calcium concentration is reached. ARVC are plated on cell culture dishes. After refreshing the cell culture medium, ARVC can be cultivated for up to six days without changing the serum-containing culture medium. Isolation of ARVC is a calcium sensitive process. Small changes in the intracellular calcium concentration cause a decrease in the quality and viability of the isolated cells.
Freshly isolated ARVC are rod shaped. Within the first days of cultivation they lose the rod-shaped morphology and form pseudopodia-like structures (spreading). During this morphological formation ARVC initially degrade their contractile elements followed by a reformation through actin stress fibers and de novo sarcomerogenesis. After one week of cultivation, most ARVC show a widespread appearance with a clearly detectable cross striation. This process is sensitive to intracellular calcium concentration, as treatment with ionomycin attenuates spreading. Key markers in this process of de- and re-differentiation are &#946;-myosin heavy chain (&#946;-MHC), oncostatin M (OSM), and swiprosin-1 (EFHD2). Recent studies have suggested that cardiac re- and de-differentiation occurring under culture conditions mimics features seen in vivo during cardiac remodeling. Therefore, isolation and cultivation of ARVC play a key role in understanding the biology of cardiomyocytes.

Here, we present a protocol for the isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC). Isolated ARVC can be used for short and long-term cultivation. The isolation and cultivation of ARVC can play a key role in developing new treatment regimens for cardiac diseases.

Isolamento e coltura dei cardiomiociti di ratto adulto.

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

Sviluppo e caratterizzazione funzionale delle cellule dendritiche tollerogeniche murino

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

Generation of Ventricular-Like HiPSC-Derived Cardiomyocytes and High-Quality Cell Preparations for Calcium Handling Characterization

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) handling and signaling pathways as well as high-throughput drug screening and toxicity assays. Herein, we provide a detailed description of the methodologies used to generate high-quality iPSC-CMs that can consistently reproduce molecular and functional characteristics across different cell lines. Additionally, a method is described to reliably assess their functional characterization through the evaluation of Ca2+ handling properties. Low oxygen (O2) conditions, lactate selection, and prolonged time in culture produce high-purity and high-quality ventricular-like cardiomyocytes. Similar to isolated adult rat cardiomyocytes (ARCMs), 3-month-old iPSC-CMs exhibit higher Ca2+ amplitude, faster rate of Ca2+ reuptake (decay-tau), and a positive lusitropic response to &#946;-adrenergic stimulation compared to day 30 iPSC-CMs. The strategy is technically simple, cost-effective, and reproducible. It provides a robust platform to model cardiac disease and for the large-scale drug screening to target Ca2+ handling proteins.

Here we describe and validate a method to consistently generate robust human induced pluripotent stem cell-derived cardiomyocytes and characterize their function. These techniques may help in developing mechanistic insight into signaling pathways, provide a platform for large-scale drug screening, and reliably model cardiac diseases.

Generazione di Cardiomiociti Ventricular-Like HiPSC-Derived e preparati cellulari di alta qualità per la caratterizzazione della movimentazione del calcio

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) ...

Functional Characterization of Endogenously Expressed Human RYR1 Variants

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia susceptibility, core myopathies and centronuclear myopathy. Because of the diverse phenotypes linked to RYR1 mutations it is fundamental to characterize their functional effects to classify variants carried by patients for future therapeutic interventions and identify non-pathogenic variants. Many laboratories have been interested in developing methods to functionally characterize RYR1 mutations expressed in patients&#39; cells. This approach has numerous advantages, including: mutations are endogenously expressed, RyR1 is not over-expressed, use of heterologous RyR1 expressing cells is avoided. However, since patients may present mutations in different genes aside RYR1, it is important to compare results from biological material from individuals harboring the same mutation, with different genetic backgrounds. The present manuscript describes methods developed to study the functional effects of endogenously expressed RYR1 variants in: (a) Epstein Barr virus immortalized human B-lymphocytes and (b) satellite cells derived from muscle biopsies and differentiated into myotubes. Changes in the intracellular calcium concentration triggered by the addition of a pharmacological RyR1 activators are then monitored. The selected cell type is loaded with a ratiometric fluorescent calcium indicator and intracellular [Ca2+] changes are monitored either at the single cell level by fluorescence microscopy or in cell populations using a spectrofluorometer. The resting [Ca2+], agonist dose response curves are then compared between cells from healthy controls and patients harboring RYR1 variants leading to insight into the functional effect of a given variant.

Here methods used to study the functional effect of RYR1 mutations endogenously expressed in Epstein Barr Virus immortalized human B-lymphocytes and muscle biopsy derived satellite cells differentiated into myotubes are described.

Caratterizzazione funzionale di varianti RYR1 umane espresse endogenamente

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia ...

Modifications of the Langendorff Method for Simultaneous Isolation of Atrial and Ventricular Myocytes from Adult Mice

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction and electrical activity. Hence, isolating viable, high-quality cardiomyocytes from the heart is the initial and most crucial experimental step. Comparing the various protocols for isolating the cardiomyocytes of adult mice, the Langendorff retrograde perfusion is the most successful and reproducible method reported in the literature, especially for isolating ventricular myocytes. However, isolating quality atrial myocytes from the perfused heart remains challenging, and few successful isolation reports are available. Solving this complicated problem is extremely important because apart from ventricular disease, atrial disease accounts for a large part of heart diseases. Therefore, further investigations on the cellular level to reveal the mechanisms are warranted. In this paper, a protocol based on the Langendorff retrograde perfusion method is introduced and some modifications in the depth of aorta cannulation and the steps that may affect the digestion process to isolate atrial and ventricular myocytes were simultaneously made. Moreover, the isolated cardiomyocytes are confirmed to be amenable to patch clamp investigation.

This protocol describes modifications of the Langendorff method including the depth of aorta cannulation for simultaneous Isolation of atrial and ventricular myocytes from adult mice.

Modifiche del metodo Langendorff per l'isolamento simultaneo di miociti atriali e ventricolari da topi adulti

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction ...