This work was supported in part by the US National Institutes of Health (R01 HL 111600) and the American Heart Association (12SDG9010015).

Alle Verfahren, die Tiere wurden von der Animal Care und Verwenden Committee der University of California, Davis zugelassen und auf den Leitfaden für die Pflege und Verwendung von Labortieren durch die National Institutes of Health veröffentlicht geklebt. 1. Vorbereitung <ol><li> Bereiten Sie zwei konzentriert (25x) Aktien von modifizierten Tyrode-Lösung im Voraus und bei 4 ° C: (1) Auf I (in mM: NaCl 3205, CaCl 2 32,5, KCl 117,5, NaH 2 PO 4 29.75, MgCl 2 26.25) und (2) Stock II (in mM: NaHCO 3 500). <ol><li> Frisch herzustellen 2 l Tyrode-Lösung der folgenden Zusammensetzung (in mM): NaCl 128,2, CaCl 2 1,3, KCl 4,7, MgCl 2 1.05, NaH 2 PO 4 1,19, NaHCO 3 20 und Glucose 11,1 durch Kombination von 1840 ml entionisiertem (DI) Wasser, 80 ml Lizenz I, 80 ml Lizenz II und 4 g Glucose. Nicht direkt Mischung Stock I und Vektor II; hinzufügenm bis 1.840 ml DI, um die Ausfällung zu vermeiden. </li></ol></li><li> Bereiten Stammlösung der Erregung und Kontraktion Entkoppler blebbistatin bei 10 mg / ml in wasserfreien Di-Dimethylsulfoxid (DMSO) im Voraus und lagern Sie die Stammlösung bei 4 ° C. </li><li> Stammlösungen von fluoreszierenden Farbstoffen: (1) spannungsempfindlichen Farbstoff RH237 (1 mg / ml in DMSO) und (2) von geringer Affinität Calciumindikator Fluo-5N AM (2 mg / ml in DMSO) gemäß den Empfehlungen des Herstellers. Mit den Vorbereitungen sofort zur Zersetzung mit anschließendem Verlust von Ladekapazität zu vermeiden. </li><li> Prime das Rezirkulieren Langendorff-Perfusionssystem mit oxygeniertem (95% O 2, 5% CO 2) Tyrodelösung. Einstellen der Strömung von O 2 / CO 2, um den pH-Wert der Tyrodelösung bei 7,4 ± 0,05 zu halten. Verwenden Sie eine in-line Nylon gewebt Nettofilter (Porengröße: 11 &amp; mgr; m), um kontinuierlich filtern das Perfusat. <ol><li> Prime das Perfusionssystem bei RT, wie nachfolgende loading Fluo-5N Uhr bei RT durchgeführt. Für Fluo-5N AM Beladung mit einem kleinen Kreislaufvolumen Perfusat (~ 150 ml). Nach der Farbstoffbeladung, verwenden Sie ein größeres Volumen an Perfusat (1 - 2 L, siehe Abbildung 1C). </li></ol></li><li> Schalten Sie Datenerfassungssystem und die Vorbereitungen für die kontinuierliche Überwachung von EKG und Perfusionsdruck. Bereiten Druck-Überwachungssystem: Verbinden Sie einen Druckwandler, um die Perfusion Linie und der Perfusionsdruck mit einem Transbridge Verstärker zu überwachen. Können die Grundlinien Perfusionsdruck auf 0 mmHg, wenn das Herz nicht in das Perfusionssystem angebracht ist. </li><li> Richten Sie die optische Abbildungskameras, um räumliche Ausrichtung zwischen dem V m und SR Ca 2+ Signale zu gewährleisten. <ol><li> Zunächst konzentrieren sich die Kameras, indem Sie ein Lineal oder ein Objekt mit Text in die Perfusion Gericht. Drehen Sie die Mapping-Kameras in eine Live View-Modus, und stellen Sie den Fokus, bis der Text ist scharf und klar. Erwerben Sie einen einzelnen Frame Bild von jeder Kamera. </li><li> OverlayDiese Bilder und die Transparenz anzupassen des oberen Bildes, so dass die beiden Bilder sichtbar sind. Der Text in den Bildern sollte genau überlappen. Wenn nicht perfekt ausgerichtet sind, kann der Winkel des dichroitischen Spiegels oder der Position jeder Kamera einzustellen, bis die beiden Bilder ausgerichtet sind. </li></ol></li></ol> 2. Ernten, Perfusion und Farbstoffbeladung von Kaninchen-Herz <ol><li> Sichern Sie das Kaninchen in einem zugelassenen Rückhalte Kaninchen. Tief anästhesiert über eine intravenöse Injektion (marginale Ohrvene) von Natriumpentobarbital (50 mg / kg) und Heparin (1000 IU). <ol><li> Als die Kaninchen Displays fehlt der nozizeptiven Reflexen, stellen Sie eine Mittellinie Hautschnitt, um den Brustbein und Rippen freizulegen. Schnitt durch das Brustbein mit stumpfen Spitze chirurgische Scheren aus dem Schwertfortsatz, um den Hammergriff. Achten Sie darauf, das Herz schädigen beim Öffnen des Brustbeins. Verbreiten Sie die Rippen, um das Herz freizulegen. </li><li> Schnell auszuschneiden das gesamte Herz-Lungen-Block durch schnelles Schneiden aller Gefäße und Bindegewebe. Immediadig legen Sie die Herz-Lungen-Block in Lösung eiskalten Tyrodes. </li></ol></li><li> Suchen Sie die Aorta für die retrograde Perfusion. Schneiden Sie den aufsteigenden Aorta proximal der drei Zweige des Aortenbogens. Kanülieren die Aorta in eine 8 G Kanüle in das Perfusionssystem verbunden. Verwenden Sie ein Stück USP 0 Seidenfaden, um die Aorta auf die Kanüle zu sichern. </li><li> Sorgfältig sezieren die Luftröhre, Lunge und epikardialen Fett aus dem Herzen. Mit einem scharfen Pinzette und Schere Dissektion, suchen Sie die Mitralklappe und sorgfältig beschädigen oder entfernen Sie eine Broschüre zur Lösung Staus in der linken Herzkammer zu verhindern. </li><li> Tauchen Sie die Herzen in der Glasmantel Perfusionskammer horizontal mit der anterioren Oberfläche des Herzens nach oben für die Bildgebung. Sichern die Kanüle in einem Stück Sylgard mit U-förmigen Stifte auf dem Silizium Boden der Schale, um die Bewegung des Herzens während der Zuordnung (Figur 1D) zu verhindern. Falls gewünscht, legen Sie ein kleines Insekt Stift an der Spitze des Herzens für stasierung. </li><li> Position Elektrokardiogramm (EKG) Elektroden, die in dem Bad an der rechten und linken Seite des Herzens. Vollständig versenken die Elektroden im Bad und sicherzustellen, dass sie nicht in Kontakt mit der Herzoberfläche, um ein Volumen-EKG durchgeführt analog zu einem Lead I Konfiguration bereitzustellen. Stellen Sie sicher, eine normale Ableitung I EKG-Morphologie. Einstellen der Strömung (25 - 35 ml / min), um sicherzustellen, dass der Aortendruck wird bei 60 gehalten - 70 mmHg. </li><li> Schalten Sie die Raumbeleuchtung und fügen Sie 0,3 bis 0,6 ml blebbistatin Stammlösung (Schritt 1.2) zum Perfusat (Endkonzentration 10 - 20 &amp; mgr; M). <ol><li> Schalten Sie die Raumbeleuchtung zu Photoinaktivierung blebbistatin vermeiden. Falls erforderlich, kann ein kleines Spotlight oder Stirnlampe verwendet werden, um Arbeitsplatzbeleuchtung bereitzustellen. Hinweis: blebbistatin ist ein Myosin-ATPase-Inhibitor und eine Erregung und Kontraktion Entkoppler. Weil Fluo-5N Lade erweiterten (60 min) RT (Hypothermie) Bedingungen erfordert (siehe Schritte 2,8-2,10), Herz-Energieproduktion beeinträchtigt sein kann. Therefore, Zugabe von blebbistatin in das Perfusat vor dem Laden Farbstoff kann den Energiebedarf 16 Verringerung und Beseitigung von Bewegungsartefakten bei der nachfolgenden optischen Aufnahmen 17. </li></ol></li><li> Bei Kontraktion des Herzens aufgehört hat (10 bis 15 min, auf der Aortendruck Aufzeichnung überprüft), um kleinvolumigen rezirkulierenden Perfusion (siehe Schritt 1.4.1) wechseln. </li><li> Bereiten Sie und laden Fluo-5N AM Beladungslösung: In 0,25 ml Fluo-5N AM-Stammlösung (Schritt 1.3) zu 0,25 ml warmem 20% Pluronic F127. 0,5 ml warmem Tyrodelösung zu der Mischung, gut mischen, und fügen Sie der kleinvolumigen Umlauf Perfusionssystem. </li><li> Kontinuierlich Perfusionsdruck und EKG-Überwachung und den Fluss ggf. einstellen. Farbstoffbeladung dauert ca. 1 h bei RT. 45 Minuten nach dem Laden begonnen hat, schalten Sie den zirkulierenden Wasserbad Erwärmung der Perfusionssystem und Perfusat auf 37 ° C zu beginnen. </li><li> Nach 60 min Fluo-5N AM Laden, switch die Perfusion des größeren Volumens Umlauf Perfusionssystem (siehe Schritt 1.4). </li><li> Verdünnte 50 ul spannungsempfindlichen Farbstoff RH237 Stammlösung (Stufe 1.3) in 1 ml warmem Tyrodelösung und langsam (über ~ 5 min) in eine Injektionsöffnung proximal der Kanüle. </li></ol> 3. Optische Mapping <ol><li> Während der letzten Momente der Farbstoffbeladung, positionieren Sie den Herzen und konzentrieren sich die optischen Abbildungskameras, um die entsprechende Sichtfeld für das Experiment zu gewährleisten. </li><li> Falls gewünscht, eine bipolare Schrittmacherelektrode an der epikardialen Oberfläche des Herzens zur Stimulierung. </li><li> Legen Sie eine Kunststoff- oder Glasdeckglas auf der Oberfläche der Perfusionskammer, um Bewegungsartefakte auf der Flüssigkeitsoberfläche, die vorhanden sein können, aufgrund von Rückführung des Perfusat zu reduzieren. Hinweis: Als Alternative zu einem Deckglas mit einem sauberen Plastik 50 mm Petrischale Cover. </li><li> Fokus der Lichtleiter gleichmäßig die Oberfläche des Herzens mit e beleuchtenxcitation Licht. Verwenden blauen Leuchtdiode (LED) Lichtquellen und das Licht weiter zu filtern mit einem 475-495 nm Bandpassfilter (Abbildung 1A). </li><li> Sammeln emittiert Fluoreszenz mit einer Makroskops und zwei komplementären Metall-Oxid-Halbleiter (CMOS) optische Abbildungskameras. Teilen Sie das emittierte Licht mit einer dichroitischen Spiegel bei 545 nm. <ol><li> Langpaßfilter langwelligeren Gruppierung, enthaltend die V m Signal bei 700 nm. Bandpassfilter die kürzeren Wellenlängen-Einheit, mit dem SR Ca 2+ Signal, 502 bis 534 nm (Abbildung 1A). Stellen Sie die Frequenz des optischen Datenerfassung, um 0,5 bis 1 kHz. </li></ol></li><li> Für jeden optischen Aufzeichnungs zunächst initiieren die gewünschte Stimulationsprotokoll, schalten Sie das Anregungslicht, und sammeln 1-4 sec von Daten. Falls gewünscht, synchronisieren die Stimulationsprotokolle und Datenerfassung. Anmerkung: Es ist nicht möglich, wieder laden, Fluo-5N AM, also das Signal-Rausch-Verhältnis (SNR) wird decrease während des Versuchs infolge einer Leckage aus dem SR färben. Begrenzen experimentelle Protokoll auf 1 - 2 h, hohe SNR-Werte zu gewährleisten. </li><li> Nach dem Versuch, wäscht den Perfusionssystem in der Sequenz von DI-Wasser, 70% Reagensalkohol und nochmals mit DI-Wasser. </li><li> Filtern jedes Datensatzes mit einer Orts Gaußfilter (Radius 3 Pixel) unter Verwendung von im Handel erhältlichen Software-Paketen gemäß dem Protokoll des Herstellers. </li><li> Falls erforderlich, räumlich ausrichten V m und SR Ca 2+ Datensätze. Wie in Schritt 1.6, Overlay-Bilder von jeder Kamera, und überprüfen Sie, dass die anatomischen Strukturen des Herzens (dh., Koronargefäße, Kanten der Atrien oder Ventrikel) oder andere Gegenstände in das Blickfeld (dh Kanüle, Schrittmacherelektrode) genau, überlappen. Falls nicht, wird räumliche Ausrichtung der Datensätze notwendig. </li><li> Schieben Sie die Top-transparentes Bild in der x- und y-Richtung, bis genaue Überdeckung erreicht wird, unter Nennung der Anzahl der Pixelmuss das Bild in jeder Richtung verschoben werden. Die gesamte Datenmenge entsprechend dem oberen Bild (entweder m oder V SR Ca 2+) ist dann bestimmt durch die Anzahl von Pixeln in jeder Richtung verschoben werden, um eine genaue Ausrichtung zwischen dem V m und SR Ca 2+ Datensätze zu gewährleisten. </li></ol>

<ol>
	<li>Herron, T. J., Lee, P., Jalife, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+voltage+and+calcium+in+cardiac+cells.">Optical imaging of voltage and calcium in cardiac cells.</a> tissues. Circulation research. 110, 609-623 (2012).</li><li>Efimov, I. R., Nikolski, V. P., Salama, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+the+heart.">Optical imaging of the heart.</a> Circulation research. 95, 21-33 (2004).</li><li>Lou, Q., Li, W., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparametric+optical+mapping+of+the+Langendorff-perfused+rabbit+heart.">Multiparametric optical mapping of the Langendorff-perfused rabbit heart.</a> Journal of visualized experiments : JoVE. , (2011).</li><li>Choi, B. R., Salama, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+maps+of+optical+action+potentials+and+calcium+transients+in+guinea-pig+hearts:+mechanisms+underlying+concordant+alternans.">Simultaneous maps of optical action potentials and calcium transients in guinea-pig hearts: mechanisms underlying concordant alternans.</a> The Journal of physiology. 529 (Pt 1), 171-188 (2000).</li><li>Kong, W., Fast, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+dye+affinity+in+optical+measurements+of+Cai(2+)+transients+in+cardiac+muscle.+American+journal+of+physiology.">The role of dye affinity in optical measurements of Cai(2+) transients in cardiac muscle. American journal of physiology.</a> Heart and circulatory physiology. 307, H73-H79 (2014).</li><li>Bers, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Excitation-Contraction Coupling and Cardiac Contractile Force. , (2001).</li><li>Bers, D. M., Shannon, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+movements+inside+the+sarcoplasmic+reticulum+of+cardiac+myocytes.">Calcium movements inside the sarcoplasmic reticulum of cardiac myocytes.</a> Journal of molecular and cellular cardiology. , (2013).</li><li>Knollmann, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+roles+of+calsequestrin+and+triadin+in+cardiac+muscle.">New roles of calsequestrin and triadin in cardiac muscle.</a> The Journal of physiology. 587, 3081-3087 (2009).</li><li>Chen, H., 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=Mechanism+of+calsequestrin+regulation+of+single+cardiac+ryanodine+receptor+in+normal+and+pathological+conditions.">Mechanism of calsequestrin regulation of single cardiac ryanodine receptor in normal and pathological conditions.</a> The Journal of general physiology. 142, 127-136 (2013).</li><li>Diaz, M. E., O'Neill, S. C., Eisner, 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=Sarcoplasmic+reticulum+calcium+content+fluctuation+is+the+key+to+cardiac+alternans.">Sarcoplasmic reticulum calcium content fluctuation is the key to cardiac alternans.</a> Circulation research. 94, 650-656 (2004).</li><li>Shannon, T. R., Guo, T., Bers, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++scraps:+local+depletions+of+free+[Ca2+]+in+cardiac+sarcoplasmic+reticulum+during+contractions+leave+substantial+Ca2++reserve.">Ca2+ scraps: local depletions of free [Ca2+] in cardiac sarcoplasmic reticulum during contractions leave substantial Ca2+ reserve.</a> Circulation research. 93, 40-45 (2003).</li><li>Kornyeyev, D., Reyes, M., Escobar, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Luminal+Ca(2+)+content+regulates+intracellular+Ca(2+)+release+in+subepicardial+myocytes+of+intact+beating+mouse+hearts:+effect+of+exogenous+buffers.">Luminal Ca(2+) content regulates intracellular Ca(2+) release in subepicardial myocytes of intact beating mouse hearts: effect of exogenous buffers.</a> American journal of physiology. Heart and circulatory physiology. 298, H2138-H2153 (2010).</li><li>Picht, E., DeSantiago, J., Blatter, L. A., Bers, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+alternans+do+not+rely+on+diastolic+sarcoplasmic+reticulum+calcium+content+fluctuations.">Cardiac alternans do not rely on diastolic sarcoplasmic reticulum calcium content fluctuations.</a> Circulation research. 99, 740-748 (2006).</li><li>Shkryl, V. M., Maxwell, J. T., Domeier, T. L., Blatter, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refractoriness+of+sarcoplasmic+reticulum+Ca2++release+determines+Ca2++alternans+in+atrial+myocytes.+American+journal+of+physiology.">Refractoriness of sarcoplasmic reticulum Ca2+ release determines Ca2+ alternans in atrial myocytes. American journal of physiology.</a> Heart and circulatory physiology. 302, H2310-H2320 (2012).</li><li>Wang, 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=Optical+mapping+of+sarcoplasmic+reticulum+Ca2++in+the+intact+heart:+ryanodine+receptor+refractoriness+during+alternans+and+fibrillation.">Optical mapping of sarcoplasmic reticulum Ca2+ in the intact heart: ryanodine receptor refractoriness during alternans and fibrillation.</a> Circulation research. 114, 1410-1421 (2014).</li><li>Wengrowski, A. M., Kuzmiak-Glancy, S., Jaimes, R., Kay, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NADH+Changes+During+Hypoxia,+Ischemia,+and+Increased+Work+Differ+Between+Isolated+Heart+Preparations.">NADH Changes During Hypoxia, Ischemia, and Increased Work Differ Between Isolated Heart Preparations.</a> American journal of physiology. Heart and circulatory physiology. , (2013).</li><li>Fedorov, V. V., 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=Application+of+blebbistatin+as+an+excitation-contraction+uncoupler+for+electrophysiologic+study+of+rat+and+rabbit+hearts.">Application of blebbistatin as an excitation-contraction uncoupler for electrophysiologic study of rat and rabbit hearts.</a> Heart rhythm : the official journal of the Heart Rhythm Society. 4, 619-626 (2007).</li><li>Myles, R. C., Wang, L., Kang, C., Bers, D. M., Ripplinger, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+beta-adrenergic+stimulation+overcomes+source-sink+mismatch+to+generate+focal+arrhythmia.">Local beta-adrenergic stimulation overcomes source-sink mismatch to generate focal arrhythmia.</a> Circulation research. 110, 1454-1464 (2012).</li><li>Marks, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+cycling+proteins+and+heart+failure:+mechanisms+and+therapeutics.">Calcium cycling proteins and heart failure: mechanisms and therapeutics.</a> The Journal of clinical investigation. 123, 46-52 (2013).</li><li>Cutler, M. 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=Targeted+sarcoplasmic+reticulum+Ca2++ATPase+2a+gene+delivery+to+restore+electrical+stability+in+the+failing+heart.">Targeted sarcoplasmic reticulum Ca2+ ATPase 2a gene delivery to restore electrical stability in the failing heart.</a> Circulation. 126, 2095-2104 (2012).</li></ol>

The authors declare that they have no competing financial interests.

Die Schlüssel zur erfolgreichen Fluo-5N Farbstoffbeladung sind der kleinvolumige rezirkulierenden Perfusion Einrichtung, die für eine hohe Fluo-5N-Konzentration ermöglicht, ohne die Notwendigkeit für große Mengen an Farbstoff, der Länge der Ladezeit (1 h) und Ausführen des Lade bei RT. Wenn das Laden bei physiologischen Temperaturen, zellulären Enzymaktivität schnell spaltet die -AM-Tag, wenn der Farbstoff kreuzt die Zellmembran, Einfangen der Farbstoffmoleküle in das Cytosol und nicht so dass sie die Membran ...

Dual optical mapping of intracellular Ca2+ and transmembrane potential (Vm) in the intact Langendorff-perfused heart has become a mainstay of investigations in cardiac electrophysiology, including mechanisms of arrhythmia and excitation-contraction coupling1-4. This approach has provided unprecedented knowledge into normal and abnormal electrophysiology and, importantly, into the bidirectional relationship between Vm and intracellular Ca2+. However, optical mapping of intracellular Ca2+ with high-affinity fluorescent indicators (such as Rhod-2 and Fluo-4) only reports on bulk changes in intracellular Ca2+ and is unable to distinguish whether these changes are due to transmembrane Ca2+ flux, release and reuptake into intracellular stores, or in most instances, some combination of both. Furthermore, high-affinity Ca2+ indicators have slow on-off kinetics and may not accurately report rapid changes in Ca2+ concentration5.
Each action potential triggers a rise in intracellular Ca2+, known as the intracellular Ca2+ transient (CaT). In the mammalian heart, approximately 70 &#8211; 90% of the total CaT is due to release of Ca2+ from the sarcoplasmic reticulum (SR) via opening of ryanodine receptors (RyRs)6. Within the SR, approximately half of the total Ca2+ is bound to calsequestrin (CSQ) and other intra-SR buffers7, which play an important role in SR Ca2+ homeostasis8,9. The amount of free SR Ca2+ dictates the driving force for SR Ca2+ release as well as gating of RyR, and therefore has a significant impact on the intracellular CaT. Furthermore, alterations in SR Ca2+ release or reuptake can, in turn, impact Vm via the electrogenic Na+-Ca2+ exchange, which may have arrhythmogenic consequences. Therefore, in addition to the CaT, monitoring of free SR Ca2+ can provide important insights into contractile and electrophysiological dysfunction.
Over the past several years, investigators have made significant advances in the monitoring of SR Ca2+ in isolated cardiac myocytes and from a single location on the intact heart. One such method requires rapid pulses of caffeine to open RyRs and the SR Ca2+ content is then inferred or calculated from the immediate rise in intracellular Ca2+10. Another intriguing approach uses low-affinity Ca2+ indicators, such as Fluo-5N11 or Mag-Fluo412, which bind to free SR Ca2+. These indicators have dissociation constants (Kd) in the range of 10 &#8211; 400 &#956;M and therefore exhibit minimal fluorescence in the cytosol compared to the SR lumen, where the Ca2+ concentration ([Ca2+]SR) is in the millimolar range. Using low-affinity Ca2+ indicators, several aspects of SR Ca2+ cycling have been investigated at the level of the isolated myocyte, including fractional SR Ca2+ release and the mechanisms of Ca2+ alternans13,14. However, in order to fully understand the heterogeneous nature of SR Ca2+ cycling in the intact heart and the role of SR Ca2+ in spatially distinct arrhythmic phenomena, methods for imaging SR Ca2+ across the epicardial surface of the intact heart are required15.
This article describes methodology for dual optical mapping of free SR Ca2+ and Vm in the intact Langendorff-perfused rabbit heart with the low-affinity Ca2+ indicator Fluo-5N. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+.

<table><tbody><tr><td>NaCl</td><td>Fisher Scientific</td><td>S271-1</td><td>Component of Tyrode&#039;s solution</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; (2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Fisher Scientific</td><td>C79-500</td><td>Component of Tyrode&#039;s solution</td></tr><tr><td>KCl</td><td>Fisher Scientific</td><td>S217-500</td><td>Component of Tyrode&#039;s solution</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt; (6H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Fisher Scientific</td><td>M33-500</td><td>Component of Tyrode&#039;s solution</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; (H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Fisher Scientific</td><td>S369-500</td><td>Component of Tyrode&#039;s solution</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher Scientific</td><td>S233-3</td><td>Component of Tyrode&#039;s solution</td></tr><tr><td>D-Glucose</td><td>Fisher Scientific</td><td>D16-1</td><td>Component of Tyrode&#039;s solution</td></tr><tr><td>95% O&lt;sub&gt;2&lt;/sub&gt; 5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td>AirGas</td><td>carbogen</td><td>For oxygenation and pH of Tyrode&#039;s solution</td></tr><tr><td>Blebbistatin</td><td>Tocris Bioscience</td><td>1760</td><td>Excitation-contraction uncoupler</td></tr><tr><td>RH237</td><td>Biotium</td><td>61018</td><td>Voltage-sensitive dye</td></tr><tr><td>Fluo-5N AM</td><td>Invitrogen</td><td>F-26915</td><td>Low-affinity Ca&lt;sup&gt;2+&lt;/sup&gt; indicator; Alternative: Invitrogen F-14204; Loading must be performed at room temperature</td></tr><tr><td>Pluronic F127</td><td>Biotium</td><td>59004</td><td>For Ca&lt;sup&gt;2+&lt;/sup&gt; indicator loading; Warm until the solution is clear before use</td></tr><tr><td>Dimethyl sulphoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D2650</td><td>For dissolving blebbistatin and dyes</td></tr><tr><td>Filter</td><td>EMD Millipore</td><td>NY1104700</td><td>11 &amp;#956;m in-line filter</td></tr><tr><td>Pressure Transducer</td><td>WPI</td><td>BLPR2</td><td>For measuring perfusion pressure</td></tr><tr><td>Transbridge Transducer Amplifier</td><td>WPI</td><td>SYS-TBM4M</td><td>For transducing/amplifing pressure signal; PowerLab may also be used with appropriate BioAmp</td></tr><tr><td>PowerLab 26T</td><td>ADInstruments</td><td></td><td>For continuous recording of pressure and ECG signals</td></tr><tr><td>THT Macroscope</td><td>SciMedia</td><td></td><td>Macroscopic optical setup. Details: 0.63X objective (NA = 0.31), 2X condensing objective, resultant field of view = 3.1 cm x 3.1 cm, depth of focus = ~ 1.5 mm</td></tr><tr><td>MiCam Ultima-L CMOS</td><td>SciMedia</td><td></td><td>Optical mapping cameras</td></tr><tr><td>Precision LED Spot Light</td><td>Mightex</td><td>PLS-0470-030-15-S</td><td>LED light source</td></tr></tbody></table>

optical mapping of intra-sarcoplasmic reticulum ca2+ and transmembrane potential in the langendorff-perfused rabbit heart

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.

1A zeigt eine schematische Darstellung der optischen Anordnung für Dual V m und SR Ca 2+ Kartierung. Mit diesem Setup, gibt es komplette spektrale Trennung der V m und SR Ca 2+ Signale (Abbildung 1B). Ein Diagramm des Dual-Schleifen Perfusionssystem für Fluo-5N Farbstoffbeladung verwendet wird, in Figur 1C veranschaulicht. Figur 1D zeigt die horizontale Ausrichtung des Herzens in das Perfusionssystem Schale....

Watch this Scientific Journal Video about Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart at JoVE.com

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Dieser Artikel beschreibt das detaillierte Protokoll und Anlagen für die duale optische Abbildung von Transmembranpotential (V m) und freie inner Retikulum (SR) notwendig Ca 2+ in der Langendorff-perfundierten Kaninchenherzen. Dieses Verfahren ermöglicht die direkte Beobachtung und Quantifizierung von V m und SR Ca 2+ Dynamik im intakten Herzens.

Optische Kartierung der Intra-Retikulum Ca<sup&gt; 2+</sup&gt; Und Transmembranpotential in der Langendorff-perfundierten Kaninchen Herz

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a ...

optical-mapping-intra-sarcoplasmic-reticulum-ca2-transmembrane

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

9.2K Aufrufe.  University of California Davis.  Dieser Artikel beschreibt das detaillierte Protokoll und Anlagen für die duale optische Abbildung von Transmembranpotential (V m) und freie inner Retikulum (SR) notwendig Ca 2+ in der Langendorff-perfundierten Kaninchenherzen. Dieses Verfahren ermöglicht die direkte Beobachtung und Quantifizierung von V m und SR Ca 2+ Dynamik im intakten Herzens. 

Serie: Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

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

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

Multiparametrische Optical Mapping der Langendorff-perfundierten Rabbit Heart

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

Forschung

Biotechnik

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

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

Optische Mapping von Aktionspotentialen und Calcium Transienten in der Maus Herz

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

High-Resolution Endocardial and Epicardial Optical Mapping in a Sheep Model of Stretch-Induced Atrial Fibrillation

Atrial fibrillation (AF) is a complex cardiac arrhythmia with high morbidity and mortality.1,2 It is the most common sustained cardiac rhythm disturbance seen in clinical practice and its prevalence is expected to increase in the coming years.3 Increased intra-atrial pressure and dilatation have been long recognized to lead to AF,1,4 which highlights the relevance of using animal models and stretch to study AF dynamics. Understanding the mechanisms underlying AF requires visualization of the cardiac electrical waves with high spatial and temporal resolution. While high-temporal resolution can be achieved by conventional electrical mapping traditionally used in human electrophysiological studies, the small number of intra-atrial electrodes that can be used simultaneously limits the spatial resolution and precludes any detailed tracking of the electrical waves during the arrhythmia. The introduction of optical mapping in the early 90's enabled wide-field characterization of fibrillatory activity together with sub-millimeter spatial resolution in animal models5,6 and led to the identification of rapidly spinning electrical wave patterns (rotors) as the sources of the fibrillatory activity that may occur in the ventricles or the atria.7-9 Using combined time- and frequency-domain analyses of optical mapping it is possible to demonstrate discrete sites of high frequency periodic activity during AF, along with frequency gradients between left and right atrium. The region with fastest rotors activates at the highest frequency and drives the overall arrhythmia.10,11 The waves emanating from such rotor interact with either functional or anatomic obstacles in their path, resulting in the phenomenon of fibrillatory conduction.12 Mapping the endocardial surface of the posterior left atrium (PLA) allows the tracking of AF wave dynamics in the region with the highest rotor frequency. Importantly, the PLA is the region where intracavitary catheter-based ablative procedures are most successful terminating AF in patients,13 which underscores the relevance of studying AF dynamics from the interior of the left atrium. Here we describe a sheep model of acute stretch-induced AF, which resembles some of the characteristics of human paroxysmal AF. Epicardial mapping on the left atrium is complemented with endocardial mapping of the PLA using a dual-channel rigid borescope c-mounted to a CCD camera, which represents the most direct approach to visualize the patterns of activation in the most relevant region for AF maintenance.

This report provides a detailed description of the methodology and results of simultaneous endocardial and epicardial optical mapping of electrical excitation in the intact left atrium of a Langendorff-perfused sheep heart during stretch-induced atrial fibrillation.

High-Resolution Endocardial und Epikardiale Optical Mapping in ein Schaf Modell Stretch-Induced Vorhofflimmern

 Atrial fibrillation (AF) is a complex cardiac arrhythmia with high morbidity and mortality.1,2 It is the most common sustained cardiac rhythm disturbance ...

Medizin

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

Optische Abbildung von Langendorff-perfundierten Rattenherzen

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

Biologie

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

Optische Bildgebung von isolierten ventrikulären Myozyten

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.

This protocol describes the procedure for sectioning and culturing human cardiac slices for preclinical drug testing and details the use of optical mapping for recording transmembrane voltage and intracellular calcium signals simultaneously from these slices.

Präklinische Kardiale Elektrophysiologie Bewertung durch Dual Voltage und Calcium Optical Mapping of Human Organotypic Cardiac Slices

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and electrophysiological phenotype in the atria. That encourages us to utilize a genetically-engineered mouse model to elucidate the mechanism of AF. However, it is difficult to evaluate the electrophysiological properties in murine atria due to their small size. This protocol describes the electrophysiological evaluation of atria using an optical mapping system with a high temporal and spatial resolution in Langendorff perfused murine hearts. The optical mapping system is assembled with dual high-speed complementary metal oxide semiconductor cameras and high magnification objective lenses, to detect the fluorescence of a voltage-sensitive dye and Ca2+ indicator. To focus on the assessment of murine atria, optical mapping is performed with an area of 2 mm &#215; 2 mm or 10 mm x 10 mm, with a 100 &#215; 100 resolution (20 &#181;m/pixel or 100 &#181;m/pixel) and sampling rate of up to 10 kHz (0.1 ms) at maximum. A 1-French size quadripolar electrode pacing catheter is placed into the right atrium through the superior vena cava avoiding any mechanical damage to the atrium, and pacing stimulation is delivered through the catheter. An electrophysiological study is performed with programmed stimulation including constant pacing, burst pacing, and up to triple extrastimuli pacing. Under a spontaneous or pacing rhythm, the optical mapping recorded the action potential duration, activation map, conduction velocity, and Ca2+ transient individually in the right and left atria. In addition, the programmed stimulation also determines the inducibility of atrial tachyarrhythmias. Precise activation mapping is performed to identify the propagation of the excitation in the atrium during an induced atrial tachyarrhythmia. Optical mapping with a specialized setting enables a thorough electrophysiological evaluation of the atrium in murine pathological models.

This protocol describes the electrophysiological evaluation of murine atria utilizing an optical mapping system with a high temporal and spatial resolution, including dual recordings of the membrane voltage and Ca2+ transient under programmed stimulation through a specialized electrode catheter.

Elektrophysiologische Bewertung der murinen Atria mit hochauflösenden optischen Mapping

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

Optokardiographie und Elektrophysiologie Studien an Ex Vivo Langendorff-perfundierten Herzen

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

High-resolution Optical Mapping of the Mouse Sino-atrial Node

Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The development of novel pharmacological therapies to cure these patients relies on the thorough understanding of both normal physiology and pathophysiology of the SAN. Among the studies of cardiac pacemaking, the mouse SAN is widely used due to its feasibility for modifications in the expression of different genes that encode SAN ion channels or calcium handling proteins. Emerging evidence from electrophysiological and histological studies has also proved the representativeness and similarity of the mouse SAN structure and functions to larger mammals, including the presence of specialized conduction pathways from the SAN to the atrium and a complex pacemakers' hierarchy within the SAN. Recently, the technique of optical mapping has greatly facilitated the exploration and investigation of the origin of excitation and conduction within and from the mouse SAN, which in turn has extended the understanding of the SAN and benefited clinical treatments of SAN dysfunction associated diseases. In this manuscript, we have described in detail how to perform the optical mapping of the mouse SAN from the intact, Langendorff-perfused heart and from the isolated atrial preparation. This protocol is a useful tool to enhance the understanding of mouse SAN physiology and pathophysiology.

Here, we present a protocol for optical mapping of electrical activity from the mouse right atrium and especially the sino-atrial node, at a high spatial and temporal resolution.

Hochauflösende optische Abbildung der Maus sinuatrialer Knoten

Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The ...

Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts

In the heart, molecular signaling studies are usually performed in isolated myocytes. However, many pathological situations such as ischemia and arrhythmias can only be fully understood at the whole organ level. Here, we present the spectroscopic technique of local field fluorescence microscopy (LFFM) that allows the measurement of cellular signals in the intact heart. The technique is based on a combination of a Langendorff perfused heart and optical fibers to record fluorescent signals. LFFM has various applications in the field of cardiovascular physiology to study the heart under normal and pathological conditions. Multiple cardiac variables can be monitored using different fluorescent indicators. These include cytosolic [Ca2+], intra-sarcoplasmic reticulum [Ca2+] and membrane potentials. The exogenous fluorescent probes are excited and the emitted fluorescence detected with three different arrangements of LFFM epifluorescence techniques presented in this paper. The central differences among these techniques are the type of light source used for excitation and on the way the excitation light is modulated. The pulsed LFFM (PLFFM) uses laser light pulses while continuous wave LFFM (CLFFM) uses continuous laser light for excitation. Finally, light-emitting diodes (LEDs) were used as a third light source. This non-coherent arrangement is called pulsed LED fluorescence microscopy (PLEDFM).

In the heart, molecular events coordinate the electrical and contractile function of the organ. A set of local field fluorescence microscopy techniques presented here enables the recording of cellular variables in intact hearts. Identifying mechanisms defining the cardiac function is critical in understanding how the heart works under pathological situations.

Optische Kartierung der Intra-Retikulum Ca

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video