NIH R01 HL085369 verir.

1. Gelişmiş stok çözümler hazırlanması <ol><li> Tyrode çözümü (16x) iki stok çözelti deiyonize su önceden hazırlayın ve 4 saklayın ° C: <ol class="lalpha"><li> Menkul Kıymetler (119,872 g / L NaCl, 3.056 g / L CaCl 2 (2H 2 O), 5,6 g / L KCl, 2,6274 g / L NaH 2 PO 4, 3,408 g / L MgCl 2 (6H 2 O), ( Fisher Bilimsel, Fair Lawn, NJ)); </li><li> Stok II (26,88 gr / L NaHCO3 (Fisher Scientific, Fair Lawn, NJ)). </li></ol></li><li> Floresan boyalar stok çözümleri hazırlayın. Tekrarlanan dondurma ve çözme önlemek için, -20 ° C, bir deney için yeterli hem de boyaların 30 ul alikotları depolamak: <ol class="lalpha"><li> Gerilim duyarlı boya RH237 (Invitrogen, Carlsbad, CA) stok solüsyonu, 1.25 mg / ml dimetil sülfoksit (DMSO, Sigma, St Louis, MO); </li><li> Kalsiyum göstergesi Rhod 02:00 (Invitrogen, Carlsbad, CA) stok solüsyonu, DMSO içinde 1 mg / ml. </li></ol></li><li> Önceden eksitasyon-kasılma uncoupler blebbistatin stok solüsyonu hazırlayın (Bioscience Tocris, St Louis, MO, DMSO içinde 2 mg / ml) ve 4 çözünmüş blebbistatin saklamak ° C </li></ol> 2. Perfüzyon çözümleri ve deney düzeneği 7 hazırlayın <ol start="4"><li> Taze 2L Tyrode çözüm hazırlamak (128.2mM NaCl, 1.3mm CaCl 2 (2H 2 O), 4.7mM KCl, 1.05mM MgCl 2 (6H 2 O), 1.19mM NaH 2 PO 4, 20mm NaHCO 3 11.1mM D-glikoz deiyonize su, pH = 7.35 ± 0.05). Stok solüsyonu Tyrode çözümü Stok I Stok II, 125 ml, 125 ml (bir deney için yeterli) 1750 mL deiyonize su eklenir ve karıştırılır ve glikoz 4g 2L yapmak için kullanılan ediliyor. </li><li> Perfüzyon sistemleri iki pompa açın. 40 ml / dak retrograd perfüzyon için kullanılan peristaltik pompa (Peri-Star, TEFE, Sarasota, Amerika Birleşik Devletleri) ayarlayın. Superfusion için kullanılan diğer bir peristaltik pompa (Cole-Parmer Masterflex Peristalic Pompa L / S, Cole-Parmer Instrument Company, Vernon Hills, Illinois) ayarlayın ve holding rezervuar 80 ml / dk &#39;ya perfüzat geri dönmek için. </li><li> Perfüzyon sistemi 2L deiyonize su ile, sonra 30 dakika ve% 70 etanol ile yıkayın. </li><li> Tyrode çözümü tüm deiyonize su sirkülasyonunu ve 5 mikron filtre (Millipore, Billerica, MA, ABD) üzerinden geçmek, oda tahliye. +37 Için perfüzat Sıcak ° C O 2 / CO 2 (% 95 /% 5) gaz köpüren bir çözüm içine bir su ceketi ve sirkülatör (ThermoNESLAB EX7, Newtown, ABD) ve oksijen perfüzat. PH metre (Oakton Instruments, Vernon Hills, IL) ile çözeltinin pH Monitör ve 7.35 ± 0,05 pH tutmak için O 2 / CO 2 köpüren hızını ayarlayabilirsiniz. Deney sırasında izleme pH ve sıcaklık devam edin. </li><li> Çift optik eşleme cihaz yüksek uzaysal (100x100 piksel, 230 ± piksel başına 20 mikron) ve temporal (1,000-3,000 kare / sn) çözünürlük iki MICAM Ultima-L CMOS kameralar (SciMedia, Costa Mesa, CA) oluşur. Fix kalsiyum görüntüleme kameranın önünde bir band geçiren filtre (590 ± 15 nm, Thorlabs, Newton, NJ); süre, uzun bir-pass filtre (&gt; 700 nm, Thorlabs, Newton, NJ) yerleştirilmiş olması gerekir belirlenen gerilim görüntüleme kamera önünde. Kameralar dikroik ayna (635 nm kesme, Omega Optik, Brattleboro, VT) içeren bir tutucu, birbirlerine dik olarak düzenlenir. Çift kamera sahibi hemen altında kalp dikroik ayna üzerine gelen emisyon ışığı odaklanan bir lens (Nikon Nikkor 55 mm 1:1.4 235.052), yoktur. Çalışma mesafesi yaklaşık 3cm. Uyarma ışık, bir halojen lamba (Newport Oriel Instruments, Stratford, CT; SciMedia, Costa Mesa, CA) tarafından oluşturulan ve ısı filtre, obtüratör ve bant geçiren filtre (520 ± 45 nm) geçirilir. Esnek bir ışık kılavuzu hazırlanması üzerine bant geçiren filtre ışık yönlendirir ve bir deklanşör hazırlık boyaların photobleaching önlemek için sadece görüntü alımı sırasında ışığa maruz olduğunu sağlamak için kullanılır. </li><li> Pacing ve algılama için önceden hazırlayın ve Ag / AgCl 2 elektrot odasında kalp yerleştirmeden önce bunları yükleyin. Amplifikatörler ve filtreler uygun seviyelere ayarlanmış olduğundan emin olun. </li></ol> 3. , Fare kalp Hasat cannulate ve Langendorff perfüzyon kurdu <ol start="10"><li> Ve intraperitoneal heparin (100 adet), ketamin / xylazine (ksilizin, 10 mg / kg ketamin, 80mg/kg vücut ağırlığı) ile fare anestezisi. Ağrı refleksi eksikliği ile uygun bir anestezi düzeyi sağlayın. </li><li> Orta sternal insizyon sonra, hızlı kalp kaldırmak ve (% 95 O 2,% 5 CO 2) oksijenli yıkayın, sabit sıcaklığı (37 ± 1 ° C) Tyrode çözümü. </li><li> Diseksiyon mikroskop kullanarakAort hızla belirlemek ve sağ subklavian arter altında çıkan aortun boyunca temiz bir kesim yapmak. Aort kısa bir bölümü daha sonra özel bir bağlı alevlendi bir ucu ile 21 gauge kanül yaptı. 4-0 siyah örgülü ipek sütür (Cerrahi Uzmanlıkları Corporation, Reading, PA) kanül üzerine kalbi düzeltmek için kullanılır. Kanülasyon sonra, kalp retrograd perfüze ve Tyrode çözümü ile superfused. Bridge Amplifikatör TBM4M, TEFE, Sarasota, retrograd perfüzyon oranı aort basıncı 60 ile 80 mmHg (Basınç detektörler, Dünya hassas aletler Inc (TEFE), Sarasota, Amerika Birleşik Devletleri tutmak için 2-5 ml / dak aralığında ayarlanır. ABD). </li><li> Sonra kalp kanüle, akciğer, timus ve yağ dokusu disseke ve kaldırılır. </li><li> Izole kalp akışı bağlı hareket etmesini önlemek için perfüzyon odasının alt apeks (Sylgard kaplamalı) (Güzel Bilim Araçları) tutturulmuş. Sağ ve sol atriyal uzantıları da gergin ve kulakçıklar optik ölçümler için maksimum yüzey alanı sağlar odası, alt (Güzel Bilim Araçlar, Inc, Foster City, CA) tutturulmuş. Çok önemli! Küçük bir silikon tüp pulmoner venler ile sol ventrikül içine yerleştirilir ve yakındaki bağ dokusu ipek sütür ile sabittir. Bu çözüm tıkanıklık ve bir eksitasyon-kasılma uncoupler ile ventriküler kasılmaları bastırılması sonra özellikle önemlidir (bkz. Bölüm 4 adımda 19) sol ventrikül, sıkışıp perfüzat asitlenme önler. </li><li> Master-8 (AMP Instruments Ltd, Kudüs, İsrail) veya PowerLab 26T (AD Instruments, Sidney, Avustralya) tarafından oluşturulan pacing uyarılara yapmak için yapılmış özel bir elektrod kalbin yüzeyine yerleştirilir. </li><li> Küçük bir kapak cam, titreşimli bir çözüm hareket artifakı azaltmak için kalp üzerinde çözüm yüzey üzerine monte edilmiştir. </li><li> Odak kalp ikaz ışığı. Buna ek olarak, çift kamera aparatı ve maksimum çözünürlük elde edilecek, böylece kalp arasındaki mesafeyi ayarlamak. </li><li> Odadaki tüm ışıkları kapatın ve elektrik kayıtları PowerLab 26T kullanarak başlamak. </li></ol> 4. Yük gerilimi ve kalsiyum duyarlı boyalar ve eksitasyon-kasılma uncoupler <ol start="19"><li> Warm-up blebbistatin 0.6 ml. 0.5 mL holding rezervuar perfüzat blebbistatin karıştırın. Tyrode çözümü 1 mL blebbistatin kalan 0.1 mL seyreltilir ve kanül yakınında bulunan bir ilaç bağlantı noktası üzerinden yavaş yavaş (bir 20 dakikalık süre içinde) enjekte. Blebbistatin yavaş yavaş hareket artifakı azaltır gözlemleyin. </li><li> 1 ml Tyrode çözümü voltaj duyarlı boya RH237 stok solüsyonu 30 mcL sulandırınız ve yavaş yavaş blebbistatin aynı enjeksiyon portuna 5-7 dakika içinde enjekte. </li><li> 30 mcL kalsiyum göstergesi Rhod-02:00 1 ml Tyrode çözümü seyreltilir ve 5-10 dakika boyunca yavaş yavaş aynı enjeksiyon portu yoluyla uygulanan Pluronic F127 (Invitrogen, Carlsbad, CA, DMSO% 20 çözüm) ile karıştırılır ve 1:1 . </li><li> Blebbistatin ve hücre zarı ve sitoplazmada ulaşmak için boyalar için 5-10 dakika bekleyin. Hareket tamamen bastırıldı protokol ile devam edin. </li><li> Sürekli bütün prosedür üzerinden monitörü EKG kayıtları, kalbin normal elektrik fonksiyonunu sağlamak. </li><li> SciMedia özel yazılım (SciMedia, Costa Mesa, CA) kullanılarak floresan sinyal kayıtları başlar. </li></ol> 5. Temsilcisi sonuçları: <img alt="Şekil 1" src="/files/ftp_upload/3275/3275fig1.jpg" /> Şekil 1 perfüzyon, elektrik kayıtları ve optik haritalama için deneysel kurulum. EM = emisyon; Lp = longpass <img alt="Şekil 2" src="/files/ftp_upload/3275/3275fig2.jpg" /> Şekil 2 ventriküler pacing sırasında kaydedilen Deneysel hazırlama ve sinyal örnekleri. Sol: EKG sinyalleri 2 Ag / AgCl disk elektrotlar (En İyi) ve S1S1 stimülasyon protokolü bir örnek gösterilmiştir (Alt) toplanan. Merkezi: Langendorff fare kalp hazırlanması. Sağ: optik Temsilcisi aksiyon potansiyelleri ve kulakçıklar (Üst) ve ventriküllerin (Alt) kalsiyum geçici sinyalleri gösterilmiştir. Sarı ok (Üst), atriyal kayıtlarında görülmektedir ventriküller, gelen floresan sinyal saçılma noktaları. AG = sol ventrikül; RV = sağ ventrikül, LA = sol atriyum; RA = sağ atrium <img alt="Şekil 3" src="/files/ftp_upload/3275/3275fig3.jpg" /> Şekil 3 V m ve vahşi bir tür fare kalp ventriküller Cat Temsilcisi optik kayıtlar. A. optik kayıtları (C) görülebilmektedir siyah noktalar işaretlenmiş eşit aralıklı yerlerden bir dizi deney hazırlık. B. Örnek dizi merkezi bir konuma V m ve CAT (bkz. kutu izleme In (C)). C. V, eşit aralıklı yerlerden dizi m (mavi) ve CAT (kırmızı) . Sinyaller 3x3 binned edildi. <img alt="Şekil 4" src="/files/ftp_upload/3275/3275fig4.jpg" /> Şekil 4 Etkinleştirme harita ve iletim. A. enine (T) ve beyaz oklarla gösterilen boyuna (L) yönden vahşi bir tür fare kalp bir örnek aktivasyon haritası. B. V m sinyalleri (Üst) ve görülen üç puan dV / dt (Alt) karşılık gelen (T1, T2, T3, L1, L2, L3). <img alt="Şekil 5" src="/files/ftp_upload/3275/3275fig5.jpg" /> Şekil 5 Aksiyon potansiyeli ve kalsiyum geçici süre analizi. % 80% 80 gevşeme (CaD80) repolarizasyon (APD80) ve kalsiyum geçici süresi A. Aksiyon potansiyeli süresi haritalar kontrol koşulları altında bir kalp (Sol) ve 30 nM isoproterenol uygulama (Sağ) sonra gösterilmiştir. Ventriküller yeşil / sarı renk (Sağ) kısaltılmış isoproterenol APD80 ve CaD80 gösterir. Vahşi tip fare ventriküllerin (Üst) ve kulakçıklar (Alt) APD80 ve CaD80 B. Örnek iz.

<ol>
	<li>Efimov, I. R., Rendt, J. M., Salama, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+maps+of+intracellular+[Ca2+]i+transients+and+action-potentials+from+the+surface+of+perfused+guinea-pig+hearts.">Optical maps of intracellular [Ca2+]i transients and action-potentials from the surface of perfused guinea-pig hearts.</a> Circulation. 90, 1-1 (1994).</li><li>Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+mapping+of+repolarization+and+refractoriness+from+intact+hearts.">Optical mapping of repolarization and refractoriness from intact hearts.</a> Circulation. 90, 1469-1480 (1994).</li><li>Choi, B. R., Salama, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+maps+of+optical+action+potentials+and+calcium+transients+in+guinea-pig+hearts:+mechanisms+underlying+concordant+alternans.">Simultaneous maps of optical action potentials and calcium transients in guinea-pig hearts: mechanisms underlying concordant alternans.</a> J Physiol. 529, 171-188 (2000).</li><li>Pruvot, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+calcium+cycling+versus+restitution+in+the+mechanism+of+repolarization+alternans.">Role of calcium cycling versus restitution in the mechanism of repolarization alternans.</a> Circ Res. 94, 1083-1090 (2004).</li><li>Efimov, I. R., Nikolski, V. P., Salama, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+the+heart.">Optical imaging of the heart.</a> Circ Res. 95, 21-33 (2004).</li><li>Fast, V. G., Dhein, S., Delmoar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recording+action+potential+using+voltage+sensitive+dyes.">Recording action potential using voltage sensitive dyes.</a> Practical methods in cardiovascular research. , 233-255 (2005).</li><li>Glukhov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+K(ATP)+channel+pharmacology+in+intact+mouse+heart.">Differential K(ATP) channel pharmacology in intact mouse heart.</a> J Mol Cell Cardiol. , (2009).</li><li>Baker, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+dispersion+of+repolarization+and+refractoriness+in+transgenic+mouse+hearts+promotes+reentrant+ventricular+tachycardia.">Enhanced dispersion of repolarization and refractoriness in transgenic mouse hearts promotes reentrant ventricular tachycardia.</a> Circ Res. 86, 396-407 (2000).</li><li>Sutherland, F. J., Hearse, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolated+blood+and+perfusion+fluid+perfused+heart.">The isolated blood and perfusion fluid perfused heart.</a> Pharmacol Res. 41, 613-627 (2000).</li><li>Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virtual+electrode+polarization+in+the+far+field:+implications+for+external+defibrillation.">Virtual electrode polarization in the far field: implications for external defibrillation.</a> Am J Physiol Heart Circ Physiol. 279, 1055-1070 (2000).</li><li>Hammouda, M., Kinosita, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+coronary+circulation+in+the+isolated+heart.">The coronary circulation in the isolated heart.</a> J Physiol. 61, 615-628 (1926).</li><li>Salama, G., Hwang, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+optical+mapping+of+intracellular+free+calcium+and+action+potentials+from+Langendorff+perfused+hearts.">Simultaneous optical mapping of intracellular free calcium and action potentials from Langendorff perfused hearts.</a> Curr Protoc Cytom. 12, 17-17 (2009).</li><li>Lou, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+panoramic+imaging+of+epicardial+electrical+activity.">Quantitative panoramic imaging of epicardial electrical activity.</a> Ann Biomed Eng. 36, 1649-1658 (2008).</li><li>Salama, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+new,+long-wavelength,+voltage-sensitive+dyes+in+the+heart.">Properties of new, long-wavelength, voltage-sensitive dyes in the heart.</a> J Membr Biol. 208, 125-140 (2005).</li></ol>

Çıkar çatışması ilan etti.

Bu deneyde bir eksitasyon-kasılma uncoupler ile ventriküler kontraksiyonlar bastırılması sonra çok önemli olan küçük bir silikon tüp ekleyerek Langendorff perfüzyon yöntemi değiştirilebilir. Silikon tüp çözüm tıkanıklığı, perfüzyon çözüm asitlenme, ve sol ventrikül iskemi gelişimini önlemek için kullanılır. Fare kalp hipotermi çok duyarlı, bu nedenle, kalp boyunca sıcaklık değişimleri aksiyon potansiyeli süresi suni farklılıklar neden olacaktır. Sonuç olarak, ısıtma sistemi, deney 8 tamamının sırasında 37 ° C sabit sıcaklıkta korumak için perfüzyon odasında hayata geçirildi . Langendorff modeli kalp innervasyon korumak olmadığından, fizyolojik, sempatik ve parasempatik 9 araştırmak için perfüzat nörotransmitterlerin ekleyerek dikkate almak gerekiyor . Retrograd perfüzyon yanı sıra, kalp superfusion ek, pH ve sıcaklık gibi uygun çevresel parametrelerin korumaya yardımcı olur. Bu yöntemde, Langendorff perfüze kalp yatay olarak yerleştirilmiştir. Dikey Langendorff perfüzyon kurulum 10 de kullanılıyor olabilir, ancak biraz daha farklı bir kardiyak mekaniği 11 neden olabilir. CMOS kameraların yanı sıra, alternatif dedektörler de mevcuttur ve haritası V m ve aynı anda CAT 12 uygulanabilir. Uygulama yüksek ayıklanmasıdır çözünürlüklü CMOS kameraların kayıtları doğruluğu garanti eder; ancak, optik haritalama sinyalleri tek bir hücre değildir. Aksine, her floresan sinyal optik büyütme bağlı olarak, yüzlerce ya da binlerce hücre geliyor. Çok daha büyük bir ventrikül floresan optik saçılma atriyal sinyallerini bozabilir, bu nedenle optik kaydedilen sinyalleri dikkatli yorumlanması gereklidir. Fare preparat başka bir sınırlama kalp 13 küçük boyutu nedeniyle yüzey eğriliği ile indüklenen sinyal bozulma ve gürültü. Iletim hızı ölçümleri sadece fare kalp eğriliği değil, aynı zamanda elektrot polarite ve sanal elektrotlar değişmiş olabilir. Iletim hızı, aktivasyon anizotropi ve repolarizasyon haritalar doğruluk elde etmek için, kalp yüzeyinde kamera odaklama düzeltmek şarttır. Bu yöntemde, gerçek zamanlı EKG kayıtları, kardiyak elektrofizyoloji optik soruşturma tamamlayabilir. Gerilim duyarlı boya (RH237) ve kalsiyum göstergesi (Rhod 02:00) hızlı yanıt nedeniyle protokol, benzer uyarma, ve farklı emisyon spektrumları 3,7 kullanılır. V m ve CAT RH237 dışında ve Rhod-02:00 3 ölçmek için kullanılan boyalar alternatif kombinasyonları vardır. Büyük bir Stoke vardiya (&gt; 200 nm) ile bir gerilim romanı duyarlı boya, PGHI, çünkü PGHI ve Rhod-02:00 14 arasında emisyon dalga boylarında büyük bir ayrılık daha iyi V m ve CAT sinyalleri izin bulundu. Gelecek gelişmeler yeni flüoresan millerini, yeni görüntüleme dedektörleri gelişimi, ve geliştirilmiş görüntü işleme yazılımı üzerine odaklanabilir. Yüksek çözünürlük ve 3 boyutlu optik haritalama için yeni optik görüntüleme yöntemleri de optik haritalama 5 gelecekte önemli yön vardır .

<table border="1"><tbody><tr><td> Kimyasal </td><td> Şirket </td><td> Katalog Numarası </td></tr><tr><td> NaCl </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> S271-1 </td></tr><tr><td> CaCl 2 (2H 2 O) </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> C79-500 </td></tr><tr><td> KCl </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> S217-500 </td></tr><tr><td> MgCl 2 (6H 2 O) </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> M33-500 </td></tr><tr><td> NaH 2 PO 4 (H 2 O) </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> S369-500 </td></tr><tr><td> NaHCO 3 </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> S233-3 </td></tr><tr><td> D-glikoz </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> D16-1 </td></tr><tr><td> Blebbistatin </td><td> Bioscience Tocris Ellisville, MO </td><td> 1760 </td></tr><tr><td> RH237 </td><td> Invitrogen, Carlsbad, CA </td><td> S1109 </td></tr><tr><td> Rhod-02:00 </td><td> Invitrogen, Carlsbad, CA </td><td> R1244 </td></tr><tr><td> Pluronic F127 </td><td> Invitrogen, Carlsbad, CA </td><td> P3000MP </td></tr><tr><td> Dimetil sulphoxide (DMSO) </td><td> Sigma, St Louis, MO </td><td> D2650 </td></tr></tbody></table>

optical mapping of action potentials and calcium transients in the mouse heart

Fare kalbi, bu türün genetik mühendisliği için düşük maliyetli teknoloji varlığı nedeniyle kardiyovasküler çalışmalar için popüler bir model. Fare kalp Kardiyovasküler fizyolojik fenotipleme transmembran potansiyeli için çeşitli sondalar (V m), kalsiyum transientler (CAT) ve diğer parametreler istihdam floresan görüntüleme kullanarak kolayca yapılabilir. Tahrik olma-daralma kaplin, aksiyon potansiyeli ve hücre içi kalsiyum dinamikleri ile karakterize edilir; bu nedenle, kalp 1-4 aynı yerden aynı anda V m ve kedi Her iki harita derece önemli. Langendorff eş zamanlı optik haritalama perfüze fare kalpleri, kalp yetmezliği, aritmiler, metabolik hastalık ve diğer kalp hastalıklarının altında yatan mekanizmaları aydınlatmak için bir potansiyele sahiptir. Aktivasyon Görselleştirme, iletim hızı, aksiyon potansiyeli süresi ve sitelerin sayısız diğer parametreler hücresel düzeyde soruşturma elde edemez ama iyi optik haritalama 1,5,6 tarafından çözüldü. Bu yazıda, state-of-the-art CMOS görüntüleme teknolojisi kullanarak yüksek alansal ve zamansal çözünürlüğe sahip fare kalplerinde V m ve kedi aynı anda optik haritalama için enstrümantasyon kurulum ve deneysel koşullar mevcut Bu yöntem ile elde edilen tutarlı optik kayıtları Langendorff perfüze fare kalpleri eşzamanlı optik haritalama uygulanabilir ve güvenilir hem de göstermektedir.

Watch this Scientific Journal Video about Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart at JoVE.com

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

Bu kağıt sağlam izole Langendorff transmembran potansiyeli (Vm) ve geçici hücre içi kalsiyum (CAT) optik haritalama sırasında diseksiyon prosedürü, enstrümantal kurulum ve deneysel koşullar fare kalpleri perfüze.

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. ...

optical-mapping-action-potentials-calcium-transients-mouse

Research

JoVE Journal

Bioengineering

27.1K Görüntüleme.  Washington University in St. Louis. Bu kağıt sağlam izole Langendorff transmembran potansiyeli (Vm) ve geçici hücre içi kalsiyum (CAT) optik haritalama sırasında diseksiyon prosedürü, enstrümantal kurulum ve deneysel koşullar fare kalpleri perfüze.

Video: Optical Mapping of Action Potentials and Calcium Transients in the Mouse 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 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 ...

Tıp

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 ...

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.

Kültürlü HL-1 Atriyal miyositlerdeki Monolayer Gerilim ve Kalsiyum Çift Kanal Optik Haritalama

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

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.

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

This article describes the detailed protocol and equipment necessary for dual optical mapping of transmembrane potential (Vm) and free intra-sarcoplasmic reticulum (SR) Ca2+ in the Langendorff-perfused rabbit heart. This method allows for direct observation and quantification of Vm and SR Ca2+ dynamics in the intact heart.

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 ...

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 ...

Mesoscopic Optical Imaging of Whole Mouse Heart

Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition (fibrosis) and cellular misalignment, can affect electrical conduction, introduce electromechanical dysfunctions and, eventually, lead to arrhythmia. Current predictive models of these functional alterations are based on non-integrated and low-resolution structural information. Placing this framework on a different order of magnitude is challenging due to the inefficacy of standard imaging methods in performing high-resolution imaging in massive tissue. In this work, we describe a methodological framework that allows imaging of whole mouse hearts with micrometric resolution. The achievement of this goal has required a technological effort where advances in tissue transformation and imaging methods have been combined. First, we describe an optimized CLARITY protocol capable of transforming an intact heart into a nanoporous, hydrogel-hybridized, lipid-free form that allows high transparency and deep staining. Then, a fluorescence light-sheet microscope able to rapidly acquire images of a mesoscopic field of view (mm-scale) with the micron-scale resolution is described. Following the mesoSPIM project, the conceived microscope allows the reconstruction of the whole mouse heart with micrometric resolution in a single tomographic scan. We believe that this methodological framework will allow clarifying the involvement of the cytoarchitecture disarray in the electrical dysfunctions and pave the way for a comprehensive model that considers both the functional and structural data, thus enabling a unified investigation of the structural causes that lead to electrical and mechanical alterations after the tissue remodeling.

We report a method for mesoscopic reconstruction of the whole mouse heart by combining new advancements in tissue transformation and staining with the development of an axially scanned light-sheet microscope.

Tüm Fare Kalbinin Mezoskopik Optik Görüntülenmesi

Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition ...