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>

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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 border="0" cellpadding="0" cellspacing="0" width="956">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">NaCl</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">S271-1</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">CaCl2 (2H2O)</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">C79-500</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">KCl</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">S217-500</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">MgCl2 (6H2O)</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">M33-500</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">NaH2PO4 (H2O)</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">S369-500</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">NaHCO3</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">S233-3</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">D-Glucose</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">D16-1</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">95% O2 5% CO2</td>
			<td style="width:167px;">AirGas</td>
			<td style="width:151px;">carbogen</td>
			<td style="width:423px;">For oxygenation and pH of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Blebbistatin</td>
			<td style="width:167px;">Tocris Bioscience</td>
			<td style="width:151px;">1760</td>
			<td style="width:423px;">Excitation-contraction uncoupler</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">RH237</td>
			<td style="width:167px;">Biotium</td>
			<td style="width:151px;">61018</td>
			<td style="width:423px;">Voltage-sensitive dye</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Fluo-5N AM</td>
			<td style="width:167px;">Invitrogen</td>
			<td style="width:151px;">F-26915</td>
			<td style="width:423px;">Low-affinity Ca2+ indicator; Alternative: Invitrogen F-14204; Loading must be performed at room temperature</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Pluronic F127</td>
			<td style="width:167px;">Biotium</td>
			<td style="width:151px;">59004</td>
			<td style="width:423px;">For Ca2+ indicator loading; Warm until the solutiion is clear before use</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Dimethyl sulphoxide (DMSO)</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:151px;">D2650</td>
			<td style="width:423px;">For dissolving blebbistatin and dyes</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Filter</td>
			<td style="width:167px;">EMD Millipore</td>
			<td style="width:151px;">NY1104700</td>
			<td style="width:423px;">11um in-line filter</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Pressure Transducer</td>
			<td style="width:167px;">WPI</td>
			<td style="width:151px;">BLPR2</td>
			<td style="width:423px;">For measuring perfusion pressure</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Transbridge Transducer Amplifier</td>
			<td style="width:167px;">WPI</td>
			<td style="width:151px;">SYS-TBM4M</td>
			<td style="width:423px;">For transducing/amplifing pressure signal; PowerLab may also be used with appropriate BioAmp</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">PowerLab 26T</td>
			<td style="width:167px;">ADInstruments</td>
			<td style="width:151px;"></td>
			<td style="width:423px;">For continuous recording of pressure and ECG signals</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">THT Macroscope</td>
			<td style="width:167px;">SciMedia</td>
			<td style="width:151px;"></td>
			<td style="width:423px;">Macroscopic optical setup. Details: 0.63x objective (NA=0.31), 2x condensing objective, resultant field of view = 3.1x3.1 cm, depth of focus = ~1.5mm</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">MiCam Ultima-L CMOS</td>
			<td style="width:167px;">SciMedia</td>
			<td style="width:151px;"></td>
			<td style="width:423px;">Optical mapping cameras</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Precision LED Spot Light</td>
			<td style="width:167px;">Mightex</td>
			<td style="width:151px;">PLS-0470-030-15-S</td>
			<td style="width:423px;">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

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

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Richtige Pflege und Reinigung des Mikroskops

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

Forschung

Biologie

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 ...

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

Semi-automatische optische Heartbeat-Analyse von kleinen Herzen

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Vorbereitung der Embryonen für Elektronenmikroskopie der Drosophila Embryonale Herz Rohr

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

Die Quantifizierung der γH2AX Foci in Response to ionisierender Strahlung

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &amp;#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

Auswertung der räumlichen Verteilung der γH2AX folgenden ionisierender Strahlung

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Klonogene Test: adhärenter Zellen

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

Visualisierung des endoplasmatischen Reticulum Subdomains in kultivierten Zellen

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte monolayers. HL-1 cells have been widely used as a 2-Dimensional cellular model for studying diverse aspects of cardiac physiology. However, it has been a great challenge to optically map calcium (Ca) transients and action potentials simultaneously from the same field of view in a cultured HL-1 atrial cell monolayer. This is because special handling and care is required to prepare healthy cells that can be electrically captured and optically mapped. Therefore, we have developed an optimal working protocol for dual channel optical mapping. In this manuscript, we have described in detail how to perform the dual channel optical mapping experiment. This protocol is a useful tool to enhance the understanding of action potential propagation and Ca kinetics in arrhythmia development.

This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

Spannung und Calcium Dual Channel Optical Mapping von Cultured HL-1 Vorhofmuskelzellen Monolayer

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte ...

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.