Name: optical mapping of intra-sarcoplasmic reticulum ca2+ and transmembrane potential in the langendorff-perfused rabbit heart
Uploaded: 2015-09-10T15:00:00
Description: 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

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

Alla som deltar i djurförsök har godkänts av Animal Care och användning kommittén vid University of California, Davis, och anslöt sig till Guide för skötsel och användning av försöksdjur publiceras av National Institutes of Health. 1. Förberedelse <ol><li> Bered två koncentrerade (25X) lager av modifierad Tyrodes lösning i förväg och förvara vid 4 ° C: (1) Lager I (i mM: NaCl 3205, CaCl2 32,5, KCl 117,5, NaH 2 PO 4 29,75, MgCl2 ...

<ol>
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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. 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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>

Nycklarna till en framgångsrik Fluo-5N färgämne lastning är den lilla volymen recirkulerande perfusion setup, vilket möjliggör en hög Fluo-5N koncentrationen utan behov av stora mängder färg, hur lång laddningstid (1 tim), och utför lastning vid RT. Om lastningen sker vid fysiologiska temperaturer, cellulär enzymatisk aktivitet snabbt klyver -AM tag när färgen korsar cellmembranet, fånga färgämnesmolekylerna i cytosolen och inte låta dem passera SR membranet. Vid RT emellertid enzymatisk aktivitet sakt...

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.

Figur 1A visar ett schematiskt diagram av den optiska konfigurationen för SR Ca 2 + kartläggning dubbla Vm och. Med denna inställning, det är klar spektral separation av V m och SR Ca 2 + -signaler (Figur 1B). Ett diagram över den dubbla slingor perfusionssystem användes för Fluo-5N färgämne lastning illustreras i Figur 1C. Figur 1D visar den horisontella orienteringen av hjärtat i perfusion skålen. Representativa <...

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

I den här artikeln beskrivs detaljerat protokoll och utrustning som behövs för dubbla optisk kartläggning av transpotential (V m) och gratis inom sarkoplasmatiska retiklet (SR) Ca 2 + i Langendorff-perfusion kaninhjärta. Denna metod möjliggör direkt observation och kvantifiering av V m och SR Ca2 + dynamik i den intakta hjärtat.

Optisk Kartläggning av Intra-sarkoplasmatiska retiklet Ca<sup&gt; 2+</sup&gt; Och transmembranpotentialen i Langendorff-perfusion Kanin hjärta

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

Research

JoVE Journal

Biology

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

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Optical Mapping of Langendorff-perfused Rat Hearts

Optisk Kartläggning av Langendorff-perfusion Rat Hearts

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

Semi-automatisk optisk Heartbeat Analys av små hjärtan

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Beredning av embryon för elektronmikroskopi av Drosophila Embryonala hjärta tub

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

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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 ...

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

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