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

optical mapping of intra-sarcoplasmic reticulum ca2+ and transmembrane potential in the langendorff-perfused rabbit heart

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.

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

Research

JoVE Journal

Biology

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

9.1K 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

Proper Care and Cleaning of the Microscope

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

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

顕微鏡の適切な手入れとクリーニング

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

生物学

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 ...

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

の心臓の鼓動の可視化ショウジョウバエ</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Semi-automated Optical Heartbeat Analysis of Small Hearts

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

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

スモールハーツの半自動光ビート解析

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

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

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

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

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

の電子顕微鏡のための胚の調製ショウジョウバエ胎児心臓の管

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

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

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

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

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

電離放射線への応答でγH2AX病巣の定量化

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

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

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

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

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

電離放射線は、次のγH2AXの空間分布の評価

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

Clonogenic Assay: Adherent Cells

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

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

クローン原性アッセイ：付着細胞

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

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.

培養HL-1心房筋細胞単層の電圧とカルシウムデュアルチャネル光学マッピング

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

High-resolution Optical Mapping of the Mouse Sino-atrial Node

Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The development of novel pharmacological therapies to cure these patients relies on the thorough understanding of both normal physiology and pathophysiology of the SAN. Among the studies of cardiac pacemaking, the mouse SAN is widely used due to its feasibility for modifications in the expression of different genes that encode SAN ion channels or calcium handling proteins. Emerging evidence from electrophysiological and histological studies has also proved the representativeness and similarity of the mouse SAN structure and functions to larger mammals, including the presence of specialized conduction pathways from the SAN to the atrium and a complex pacemakers' hierarchy within the SAN. Recently, the technique of optical mapping has greatly facilitated the exploration and investigation of the origin of excitation and conduction within and from the mouse SAN, which in turn has extended the understanding of the SAN and benefited clinical treatments of SAN dysfunction associated diseases. In this manuscript, we have described in detail how to perform the optical mapping of the mouse SAN from the intact, Langendorff-perfused heart and from the isolated atrial preparation. This protocol is a useful tool to enhance the understanding of mouse SAN physiology and pathophysiology.

Here, we present a protocol for optical mapping of electrical activity from the mouse right atrium and especially the sino-atrial node, at a high spatial and temporal resolution.