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

Hayvanları da içeren tüm prosedürler California, Davis Üniversitesi Hayvan Bakım ve Kullanım Kurulu tarafından onaylanmış ve Ulusal Sağlık Enstitüleri tarafından yayınlanan Laboratuvar Hayvanları Bakım ve Kullanım Kılavuzu yapıştırılır bulundu. 1. Hazırlık <ol><li> 4 ° C&#39;de önceden ve mağaza modifiye Tyrode çözeltisi iki konsantre edildi (25X) stokları hazırlayın: (1) Stok I (mM olarak: NaCl 3205, CaCl2 32.5, KCI 117.5, NaH 2 PO 4 29,75, MgCI2 26,25) ve (2) (mM olarak: 500 NaHCO 3) Stok II. <ol><li> (DI) NaCl 128.2, CaCl2 1.3, KCI 4.7, MgCl2 1.05, NaH 2 PO 4 1,19, NaHCO 3 20 ve deiyonize 1840 ml birleştirerek glikoz 11.1: Taze (mM olarak) aşağıdaki bileşime sahip 2 L&#39;lik Tyrode solüsyonu hazırlamak Su, 80 mi Hazır I Stok II 80 mi, ve glukoz, 4 g. Doğrudan Stok I ve II Stok karışmaz; eklemek1840 ml DI m çökmesini önlemek için. </li></ol></li><li> Önceden 10 mg susuz di-metilsülfoksit (DMSO) / ml uyanlma-kasılma uncoupler blebbistatin stok çözeltisi hazırlamak ve 4 ° C &#39;de stok solüsyonu saklayın. </li><li> Floresan boyalar stok çözelti hazırlayın: (1) voltaj duyarlı boya RH237 (DMSO içinde 1 mg / ml) ve (2) Düşük afiniteli kalsiyum göstergesi Fluo-5N AM, üreticinin tavsiyelerine göre (DMSO içinde 2 mg / ml). Yükleme kapasitesi kaybı sonucu ile, bozulmayı önlemek için, hemen hazırlıkları kullanın. </li><li> Prime oksijenli sahip çevrimli Langendorff perfüzyonu sistemi (% 95 O2,% 5 CO2), Tyrode çözeltisi. 7.4 ± 0.05 Tyrode çözümü pH tutmak için O 2 / CO 2 akışını ayarlayın. Sürekli perfüzyon filtre: bir in-line naylon dokuma ağ filtre (11 um gözenek boyutu) kullanın. <ol><li> Sonraki loadi olarak oda sıcaklığında Başbakan perfüzyon sistemi,Fluo-5N AM ng oda sıcaklığında gerçekleştirilir. Fluo-5N AM yükleme için, çevrimli perfüzat küçük bir hacim (~ 150 ml) kullanın. Boya yüklemesinin ardından, perfüzat daha büyük bir hacim kullanımı (1-2 L, Şekil 1C bakınız). </li></ol></li><li> Veri toplama sistemi açın ve EKG ve perfüzyon basıncının sürekli izlenmesi için hazırlar. Basınç izleme sistemi hazırlayın: perfüzyon hattına bir basınç transdüktörü bağlayın ve bir transbridge amplifikatörü ile perfüzyon basıncını izlemek. Kalp perfüzyon sistemine bağlı değilken 0 mmHg bazal perfüzyon basıncını ayarlayın. </li><li> V m ve SR Ca 2 + sinyalleri arasındaki uzaysal uyum sağlamak için optik haritalama kameraları aynı hizaya getirin. <ol><li> İlk olarak, bir cetvel ya da perfüzyon çanak içine metin ile bir nesne yerleştirerek kameralar odaklanır. Canlı görünüm moduna haritalama kameraları açın ve metin canlı ve net olana kadar odağı ayarlamak. Her bir kamera tek bir kare görüntü elde edin. </li><li> YerleşimiHer iki resim görünür, böylece bu görüntülerin ve üst resmin saydamlığını ayarlamak. Görüntülerdeki metin tam üst üste gelmelidir. Mükemmel hizalanmış değilse iki görüntü aynı hizada kadar, dikroik ayna veya her kameranın konumunun açısını ayarlayın. </li></ol></li></ol> Tavşan Kalbin 2. Hasat, Perfüzyon ve Boya yükleme <ol><li> Onaylanmış bir tavşan süzgeç tavşan sabitleyin. Derin sodyum pentobarbital (50 mg / kg) ve heparin (1000 IU) bir intravenöz enjeksiyon (marjinal kulak daman) ile anestetize. <ol><li> Tavşan görüntüler nosiseptif reflekslerin eksikliği olduğunda, bir orta hat cilt kesi sternum ve kaburga maruz olun. Manubrium için xiphoid gelen künt uçlu cerrahi makas ile sternum ile kesin. Sternumu açarken kalbi zarar vermemeye özen gösterin. Kalp maruz kaburga yayıldı. </li><li> Hızla hızla tüm damarları ve bağ dokusu kesim tarafından tüm kalp-akciğer bloğu tüketim. Immediately buz gibi soğuk Tyrode çözeltisi içine kalp-akciğer bloğu yerleştirin. </li></ol></li><li> Retrograd perfüzyon için aorta bulun. Aortik arkın üç şubelerine sadece proksimal asendan aort kesin. Perfüzyon sistemine bağlı bir 8 G kanül aort kanüle. Kanül üzerine aorta güvenli USP 0 ipek sütür bir parça kullanın. </li><li> Dikkatle kalp trakea, akciğerler ve epikardiyal yağ teşrih. Keskin forseps ve diseksiyon makas ile, mitral kapak bulun ve dikkatle zarar veya sol ventrikül çözüm sıkışıklığını önlemek için tek broşür kaldırın. </li><li> Görüntüleme için yukarı bakacak şekilde kalbin ön yüzeyi ile yatay cam ceketli perfüzyon odasında kalbi Batmak. Haritalama (Şekil 1D) sırasında kalbin hareket etmesini önlemek için çanağın silikon alt üzerine U-şekilli pimlerle Sylgard parçasına kanül elde edin. İsterseniz, sta için kalbin tepesinde küçük bir böcek pin eklemekbilization. </li><li> Pozisyon elektrokardiyogram (EKG) kalbin sağ ve sol tarafında banyosunda elektrotlar. Tamamen banyosunda elektrotlar daldırın ve onlar Kurşun Ben yapılandırmasına bir hacim yapılan EKG benzer sağlamak için kalp yüzeyi ile temas halinde olmadığından emin olun. Normal Kurşun I EKG morfoloji doğrulayın. 70 mmHg - aort basıncı 60 muhafaza edilmesinin sağlanması için - (35 ml / dakika 25) akışını ayarlayın. </li><li> Oda ışıkları kapatın ve 0.3 eklemek - Perfüzat (- 20 uM son konsantrasyon 10) ile blebbistatin stok solüsyonu (adım 1.2) 0.6 ml. <ol><li> Blebbistatin bir photoinactivation önlemek için oda ışıkları kapatın. Gerekirse küçük bir spot ışığı veya far görevi aydınlatma sağlamak için kullanılabilir. Not: Blebbistatin bir miyozin ATPaz inhibitörü ve bir eksitasyon-kasılma uncoupler olduğunu. , Kardiyak enerji üretimi bozulabilir - Flu-5N yükleme genişletilmiş (60 dk) RT (hipotermi) koşulları gerektirdiğinden (2.10 adımları 2.8 bakınız). Therefore, Perfüzat önce içine blebbistatin eklenmesi enerji talebini azaltmak 16 ve sonraki optik kayıtları 17 sırasında hareket eserler ortadan kaldırabilir yükleme boya. </li></ol></li><li> Kalbin kasılması bittikten sonra (10 - aort basınç kayıt doğrulandı 15 dk), küçük hacimli çevrimli perfüzyon sistemi (adım 1.4.1 bakınız) geçmek. </li><li> Hazırlayın ve Fluo-5N AM yükleme çözüm yük: 0.25 ml ılık% 20 Pluronic F127 için Fluo-5N AM stok solüsyonu (adım 1.3) 0.25 ml ekleyin. , 0,5 ml karışımına sıcak Tyrode çözeltisi ekleyin, iyice karıştırılmakta ve küçük hacimli çevrimli perfüzyon sistemine ekleyin. </li><li> Sürekli perfüzyon basıncı ve EKG izlenmesi ve gerekirse akışını ayarlamak. Boya yükleme RT&#39;de yaklaşık 1 saat sürer. Yüklemeden sonra 45 dk 37 ° C perfüzyon sistemi ve perfüzyon ısınma başlamak için dolaşan su banyosu açın başladı. </li><li> Fluo-5N AM yükleme, swi 60 dakika sonraDaha büyük hacimli sirkülasyon perfüzyon sistemine perfüzyon Tch (adım 1.4). </li><li> Sıcak Tyrode çözeltisi 1 ml voltajı duyarlı boya RH237 stok çözeltisinin (adım 1.3) 50 ul seyreltilir ve kanül, bir enjeksiyon noktası proksimal içerisine (5 ~ dakika boyunca) yavaş yavaş ekleyin. </li></ol> 3. Optik Haritalama <ol><li> Boya yükleme son anlarında kalbi konumlandırmak ve deney için görüş uygun alanı sağlamak için optik haritalama kameraları odaklanır. </li><li> Eğer arzu edilirse, kalp atışı düzenleme epikardiyal yüzeyi üzerine yerleştirilen iki kutuplu bir elektrot ilerleme hızı yerleştirin. </li><li> Bağlı perfüzat devridaim mevcut olabilir, sıvı yüzeyi üzerinde hareket paraziti azaltmak perfüzyon odasına yüzeyi üzerinde bir plastik ya da cam kapak kayma yerleştirin. Not: Bir kapak kayma bir alternatif, temiz bir plastik 50 mm Petri kabı kapağı kullanmak gibi. </li><li> Üniform e kalbin yüzeyine aydınlatmak için ışık kılavuzları Odakxcitation ışığı. 495 nm bant geçiren filtre (Şekil 1A) - Mavi ışık yayan diyot (LED) ışık kaynağı kullanın ve daha bir 475 ile ışık filtresi. </li><li> Bir macroscope ve iki tamamlayıcı metal oksit-yarıiletken (CMOS) optik haritalama kameralar ile yayılan floresan toplayın. 545 nm&#39;de dikroik ayna ile yayılan ışığı bölün. <ol><li> Uzun pas 700 nm&#39;de, V m sinyalini içeren, uzun dalga boyu parçasını filtre. 534 nm (Şekil 1A) - Bant geçiren 502 den, SR Ca 2 + sinyalini içeren, kısa dalga boylu parçasını filtre. 1 kHz - 0.5 optik veri toplama sıklığını ayarlayın. </li></ol></li><li> Her optik kayıt için, ilk olarak, istenen düzenlemeyi başlatmak ikaz ışığı açmak ve 1 toplamak - veri 4 sn. İstenirse, ilerleme hızı protokolleri ve veri toplama senkronize. Not: mümkün değildir Fluo-5N AM, bu nedenle sinyal-gürültü oranını (SNR) re-load düflüfl olacakse SR sızıntıyı boya nedeniyle deney boyunca. Yüksek SNR değerleri sağlamak için 2 saat - 1 deneysel protokol sınırlayın. </li><li> Deney sonra, Dİ su ile tekrar DI su,% 70 reaktif alkol sırayla perfüzyon sistemi yıkama ve. </li><li> Üreticinin protokolüne göre ticari yazılım paketleri kullanılarak mekansal Gauss filtre (yarıçap 3 piksel) her veri kümesi Filtre. </li><li> Gerekirse, mekansal V m ve SR Ca 2 + veri setleri aynı hizaya getirin. Adım 1.6 olduğu gibi, her kamera görüntüleri kaplaması ve doğrulayın görünümü (yani, kanül, kalp pili elektrodu) alanında kalbin anatomik yapıları (yani., Koroner damar, kulakçıklar kenarlarına veya ventriküller) ya da diğer öğeleri tam olarak örtüşmektedir. Değilse, veri setlerinin mekânsal uyum gereklidir. </li><li> Piksel sayısı not ederek, tam örtüşme kadar x ve y-yönünde elde edilir üst saydam bir görüntü ShiftResim, her yönünde kaydırılabilir olmalıdır. Üst görüntüde (V m veya SR Ca 2+ ya) karşılık gelen tüm veri kümesi sonra V m ve SR Ca 2 + veri setleri arasındaki hassas hizalama sağlamak için her yönde piksel belirlenen sayısına göre kaydırılır gerekmektedir. </li></ol>

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The authors declare that they have no competing financial interests.

Başarılı Fluo-5N boya yükleme anahtarları boyanın büyük miktarda yükleme süresi (1 saat) uzunluğu gerek kalmadan yüksek Fluo-5N konsantrasyonu sağlar küçük hacimli çevrimli perfüzyon kurulum vardır ve yükleme gerçekleştirme oda sıcaklığında karıştırıldı. Yükleme hızla fizyolojik sıcaklıklarda, hücresel enzimatik aktivite böler boya sitozolde boya moleküllerini yakalama ve onları SR zarı geçmeye izin vermiyor, hücre zarı haçlar -AM etiketi gerçekleştirilir edin. Oda sıcaklı...

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.

Şekil 1A, çift V m ve SR Ca2 + eşleme için optik konfigürasyonunda şematik bir diyagramını göstermektedir. Bu ayar ile, V m ve SR Ca 2 + sinyalleri (Şekil 1B) tam spektral ayrılık yoktur. Fluo-5N boya yüklemesinin için kullanılan çift döngü perfüzyon sistemin bir şeması Şekil 1C &#39;de gösterilmiştir. Şekil 1 D perfüzyon çanak kalp yatay yönünü gösterir. Örnek V m ve...

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

Bu makalede, Langendorff-perfüze tavşan kalbinde ayrıntılı protokol ve transmembran potansiyeli (V m) ikili optik haritalama ve ücretsiz içi sarkoplazmik retikulum (SR) için gerekli donanımın Ca 2+ açıklanır. Bu yöntem, sağlam kalbinde, doğrudan gözlem ve V m ölçümü ve SR Ca 2 + dinamikleri sağlar.

Intra-Sarkoplazmik Retikulum Ca Optik Haritalama<sup&gt; 2+</sup&gt; Ve Langendorff-perfüze Tavşan Heart Transmembran Potansiyeli

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 Görüntüleme.  University of California Davis.  Bu makalede, Langendorff-perfüze tavşan kalbinde ayrıntılı protokol ve transmembran potansiyeli (V m) ikili optik haritalama ve ücretsiz içi sarkoplazmik retikulum (SR) için gerekli donanımın Ca 2+ açıklanır. Bu yöntem, sağlam kalbinde, doğrudan gözlem ve V m ölçümü ve SR Ca 2 + dinamikleri sağlar. 

Video: 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.

Çok parametreli Langendorff perfüze Tavşan Kalp Optik Haritalama

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

Biyomühendislik

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.

Fare Kalp Aksiyon Potansiyelleri ve Kalsiyum Geçici Optik Haritalama

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.

Stretch Bağlı Atriyal Fibrilasyon Koyun Modelinde Yüksek Çözünürlüklü Endokardial ve Epikardiyal Optik Haritalama

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

Tıp

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).

Langendorff perfüze sıçan Kalpler Optik Haritalama

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

Biyoloji

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.

İzole Murine Ventriküler Miyositlerin Optik Görüntülemesi

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.

İnsan Organotipik Kardiyak Dilimlerinin Çift Voltaj ve Kalsiyum Optik Haritalaması ile Preklinik Kardiyak Elektrofizyoloji Değerlendirmesi

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.

Yüksek çözünürlüklü optik eşleştirmenin fare Atria Elektrofizyolojik değerlendirme

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.

Ex Vivo Langendorff perfüzyonlu Kalplerin Optokardiyografi ve Elektrofizyoloji Çalışmaları

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.

Fare Çin-atriyal düğüm yüksek çözünürlüklü optik Haritalama

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.

Intra-Sarkoplazmik Retikulum Ca Optik Haritalama

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