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

動物に関わるすべての手続きは、カリフォルニア大学デ​​ービス校の動物実験委員会によって承認され、国立衛生研究所によって公開実験動物の管理と使用に関する指針に付着させました。 1.準備<ol><li> 4℃に予め、店舗で修正されたタイロード液の2集中（25X）の株式を準備します（1）株式のI（MM中：NaClの3205、CaCl 2を32.5、塩化カリウム117.5、のNaH 2 PO 4 29.75、のMgCl 2 26.25）と（2）株式II（MM中：のNaHCO 3 500）。 <ol><li> NaClの128.2、 塩化カルシウム1.3、塩化カリウム4.7、MgCl 2を1.05、のNaH 2 PO 4 1.19、NaHCO 3で20、脱イオン（DI）の1840ミリリットルを組み合わせることにより、11.1グルコース：たて（MM）で、以下の組成の2 Lのタイロード溶液を調製水、80ミリリットル証券私、証券IIの80ミリリットル、およびグルコースの4グラム。直接株式私と証券IIを混在させないでください。加えます沈殿を回避するために1840ミリリットルのDIにメートル。 </li></ol></li><li>事前に無水ジメチルスルホキシド（DMSO）中に10mg / mlで興奮収縮脱共役剤のブレビスタチンの原液を調製し、4℃で保存溶液を保存します。 </li><li>蛍光色素のストック溶液を準備します（1）電位感受性色素RH237（DMSO中1mg / ml）及び（2）低親和性のカルシウム指示薬フルオ-5N AM、製造業者の推奨に従って（DMSO中に2mg / ml）を。積載量のその後の損失と分解を避けるために、すぐに準備を使用してください。 </li><li>プライム酸素（95％O 2、5％CO 2）タイロード溶液で再循環ランゲンドルフ灌流システム。 7.4±0.05でタイロード液のpHを維持するために、O 2 / CO 2の流れを調整します。継続的に灌流液をフィルタリングする：イン・ラインナイロン織りネットフィルター（11μmの孔サイズ）を使用します。 <ol><li>その後のLOADIとして内閣総理RTで灌流システム、Fluo-5N AMのNGを室温で行われます。フルオ-5N AMローディングのために、再循環灌流液の少量（〜150ミリリットル）を使用します。色素負荷後、灌流液の大量使用（1 - 2 Lを、 図1Cを参照）。 </li></ol></li><li>データ収集システムの電源をオンにし、ECGおよび灌流圧を連続的に監視するための準備。圧力監視システムを準備します。灌流ラインに圧力変換器を接続し、transbridgeアンプで灌流圧を監視します。心臓が灌流システムに接続されていないとき0 mmHgのベースライン潅流圧を調整します。 </li><li> V mとSRのCa 2+シグナルとの間の空間的な位置合わせを確実にするために、光学マッピングカメラの位置を合わせます。 <ol><li>まず、灌流皿にルーラーやテキストを使用してオブジェクトを配置することによって、カメラの焦点を合わせます。ライブビューモードにマッピングカメラを回して、テキストはくっきりと鮮明になるまでフォーカスを調整します。各カメラから単一のフレーム画像を取得します。 </li><li>オーバーレイこれらの画像との両方の画像が表示されるように、トップ画像の透明度を調整します。画像内のテキストが正確にオーバーラップする必要があります。完全に整列していない場合、2つの画像が位置合わせされるまで、ダイクロイックミラーまたは各カメラの位置の角度を調整します。 </li></ol></li></ol> 2.ウサギの心の収穫、灌流、および色素ローディング<ol><li>承認されたウサギの制止にウサギを固定します。深くペントバルビタールナトリウム（50mg / kg）およびヘパリン（千IU）の静脈内注射（辺縁耳静脈）を介して麻酔。 <ol><li>ウサギは、侵害受容反射の欠如を表示する場合は、胸骨と肋骨を露出するために正中線皮膚切開を行います。胸骨柄の剣状突起から鈍い先端の外科ハサミで胸骨を切断。胸骨を開いている間に心を傷つけないように注意してください。心臓を露出するためのリブを広げます。 </li><li>すぐに急速にすべての血管および結合組織を切断することにより、全体の心肺ブロックを切り出します。 Immediately氷冷タイロード溶液中に心肺ブロックを配置します。 </li></ol></li><li>逆行性灌流のための大動脈の位置を確認します。大動脈弓の3支店のすぐ近位上行大動脈をカットします。灌流システムに接続された8 Gカニューレに大動脈にカニューレを挿入。カニューレ上に大動脈を確保するために、USP 0絹縫合糸の一部を使用します。 </li><li>慎重に心臓から気管、肺、および心外膜脂肪を分析。鋭いピンセットと解剖ハサミで、僧帽弁を見つけて、慎重に左心室内の溶液の混雑を防ぐために、1リーフレットを損傷したり、削除します。 </li><li>イメージングのために上向きに心臓の前面と水平にガラスジャケット付き灌流チャンバーに心を沈めます。マッピング（ 図1D）の間、心臓の動きを防止するために皿のシリコン底部にU字型ピンとシルガードの片にカニューレを固定します。必要に応じて、STAのための心の頂点に小さな虫ピンを挿入bilization。 </li><li>ポジション心電図（ECG）は、心臓の右側と左側にバス内の電極。完全に浴に電極を浸し、彼らがリード私の構成に類似したボリューム行わ心電図を提供するために、心臓の表面に接触していないことを確認します。通常のリードI ECG形態を確認してください。 70 mmHgの - 大動脈圧は60に維持されることを確実にするために - （35ミリリットル/分25）の流れを調整します。 </li><li>部屋の照明をオフにして、0.3を追加 - （ - 20μMの最終濃度10）灌流液にブレビスタチン原液（ステップ1.2）の0.6ミリリットルを。 <ol><li>ブレビスタチンの光不活性化を回避するために、部屋の照明をオフにします。必要に応じて、小さなスポットライトやヘッドランプは、作業照明を提供するために使用することができます。 注意：ブレビスタチンは、ミオシンATPアーゼ阻害剤および興奮収縮脱共役剤です。フルオ-5Nの荷重が延長（60分）、RT（低体温）条件（ステップ2.8を参照してください。 - 2.10）を必要とするので、心臓のエネルギー生産が損なわれることがあります。目ereforeは、ロードを染める前に灌流液へのブレビスタチンの添加は、エネルギー需要16を削減し、その後の光学記録17の間にモーションアーチファクトを除去することができます。 </li></ol></li><li>心臓の収縮が停止した場合（10 - 大動脈圧の記録に検証15分）、小容量循環灌流システム（ステップ1.4.1を参照）に切り替えます。 </li><li>準備したFluo-5N AMのローディング溶液をロード：0.25ミリリットル暖かい20％プルロニックF127にフルオ-5N AM原液（ステップ1.3）の0.25ミリリットルを追加します。 0.5mlの混合物に暖かいタイロード液を加えよく混ぜ、および小容量循環灌流システムに追加します。 </li><li>連続灌流圧と心電図をモニターし、必要に応じて流れを調整します。色素負荷は、室温で約1時間かかります。ロードが開始された45分後、37℃に灌流システムおよび灌流液を暖め始めるために循環水浴をオンにします。 </li><li>フルオ-5N AM搭載の60分後、SWI大容量循環灌流システムに灌流をTCH（ステップ1.4を参照）。 </li><li>温かいタイロード液1mlに電位感受性色素RH237ストック溶液（ステップ1.3）、50μlの希釈し、カニューレに注入口の近位に（〜5分かけて）ゆっくりと加えます。 </li></ol> 3.光学マッピング<ol><li>色素負荷の最後の瞬間の間に、心を合わせて、実験用のビューの適切なフィールドを確保するために、光学マッピングカメラの焦点を合わせます。 </li><li>必要に応じて、ペーシングのために心臓の心外膜表面に双極ペーシング電極を配置します。 </li><li>灌流液の循環に存在することができる液面にモーションアーチファクトを低減するために灌流チャンバーの表面のプラスチックまたはガラスカバースリップを置きます。 注意：カバースリップに代わるものとして、クリーンなプラスチック製の50ミリメートルのペトリ皿のカバーを使用しています。 </li><li>均一Eで心臓の表面を照明する光ガイドをフォーカスxcitation光。 495 nmのバンドパスフィルタ（ 図1A） -青色発光ダイオード（LED）光源を使用し、さらに475の光をフィルタリングします。 </li><li>マクロスコープ及び二つの相補型金属酸化膜半導体（CMOS）光学的マッピングカメラで放出された蛍光を集めます。 545 nmのダイクロイックミラーで放出された光を分割します。 <ol><li>ロングパスは700 nmで、V mの信号を含む、より長い波長成分をフィルタ。 534ナノメートル（ 図1A） -バンドパス502から、SR の Ca 2+シグナルを含む、短波長成分をフィルタリングします。 1 kHzの - 0.5に光データ収集の頻度を設定します。 </li></ol></li><li>各光記録のために、第1の所望のペーシングプロトコルを開始し、励起光をオンにし、1収集 - データの4秒。必要に応じて、ペーシングプロトコルとデータ収集を同期させます。 注：これは、再ロードのFluo-5N AMすることは不可能であるので、信号対雑音比（SNR）がdecreaますSE、SRからの漏れを染料による実験を通して。高いSNR値を確保するために、2時間 - 1に実験プロトコルを制限します。 </li><li>実験の後、DI水で再びDI水、70％の試薬アルコールの順序で灌流システムを洗浄し、。 </li><li>製造業者のプロトコルに従って、市販のソフトウェアパッケージを使用した空間ガウスフィルタ（半径3ピクセル）と各データセットをフィルタリングします。 </li><li>必要に応じて、空間V mとSR の Ca 2+のデータセットを整列させます。ステップ1.6のように、各カメラからの画像をオーバーレイし、検証し、そのビュー（ すなわち、カニューレ、ペーシング電極）の分野における心臓の解剖学的構造（すなわち、冠血管系、心房のエッジまたは心室）、またはその他の項目を正確に重複。そうでない場合、データセットの空間的な位置合わせが必要です。 </li><li>正確なオーバーラップが達成されるまで、画素数のノートを作り、x軸とy方向に上部の透明画像をシフト画像は、それぞれの方向にシフトしなければなりません。トップ画像（V mまたはSRのCa 2+のいずれか）に対応するデータセット全体は、その後、V mとSRのCa 2+データセット間の正確な位置合わせを保証するために、各方向の画素の決定された数だけシフトする必要があります。 </li></ol>

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The authors declare that they have no competing financial interests.

成功のFluo-5Nの色素負荷への鍵は、色素の大量を必要とせずに高いのFluo-5N濃度を可能にする小容量循環灌流セットアップ、ロード時間（1時間）の長さ、およびロードを実行していますRTで。ロードが生理的温度、細胞の酵素活性で実行された場合、色素が細胞質ゾルに染料分子を捕捉し、それらは、SR膜を通過することができない、細胞膜を横切るときに迅速-AMタグを切断します。 RTで、しか...

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.

図1Aは、二重のV mおよびSR の Ca 2+マッピングの光学的構成の概略図を示します。この設定では、V mの完全なスペクトル分離とSRのCa 2+シグナル （ 図1B）があります 。フルオ-5N染料ロードに使用デュアルループ灌流システムの図を図1Cに示されている。 図1Dは 、灌流皿の中の心の水平方向を示して?...

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

この記事では、ランゲンドルフ灌流ウサギの心臓における詳細なプロトコルおよび膜貫通電位 （Vm）のデュアル光学マッピングとフリーイントラ筋小胞体（SR）のために必要な機器のCa 2+を説明しています。この方法は、無傷の中心部にあるV mとSRのCa 2+動態の直接観察と定量化を可能にします。

イントラ小胞体のCaの光学マッピング<sup&gt; 2+</sup&gt;とランゲンドルフ灌流ウサギの心臓における膜電位

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 ビュー.  University of California Davis. この記事では、ランゲンドルフ灌流ウサギの心臓における詳細なプロトコルおよび膜貫通電位 （Vm）のデュアル光学マッピングとフリーイントラ筋小胞体（SR）のために必要な機器のCa 2+を説明しています。この方法は、無傷の中心部にあるV mとSRのCa 2+動態の直接観察と定量化を可能にします。 

ビデオ: 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.

ランゲンドルフ灌流ウサギの心臓のMultiparametric光学マッピング

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

生物工学

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.

マウスの心臓の活動電位およびカルシウムトランジェントの光学的マッピング

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.

ストレッチ誘発性心房細動の羊のモデルで高解像度の心内膜と心外膜光学マッピング

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

医学

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).

ランゲンドルフ灌流ラット心臓の光学的マッピング

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

生物学

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.

単離マウス心室筋細胞の光学イメージング

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.

ヒトの神経ノチカルスライスの二重電圧およびカルシウム光学マッピングによる前臨床心臓電気生理学的評価

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.

高解像度光学マッピングとマウスの心房の電気生理学的評価

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.

元ヴィヴォ・ランゲンドルフ・パーフュード・ハーツの眼電図と電気生理学研究

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.

マウス洞房結節の高分解能光学マッピング

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.

イントラ小胞体のCaの光学マッピング

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