We are supported by the University of Wisconsin-Madison Medical School start-up (A.V.G.).

全ての実験は、実験動物の管理と使用に関する健康ガイドの国立研究所（NIHパブ号80から23）に従って行きました。これらの研究で使用されているすべての方法およびプロトコルは、（公開番号85から23は、1996年改訂）NIHによって公開実験動物の管理と使用に関するガイドライン以下のウィスコンシン大学の動物実験プロトコル委員会によって承認されています。本研究で用いた全ての動物は、実験動物の管理と使用に関する指針に準拠して人道的な世話を受けました。 1.ハートの取り外しおよびランゲンドルフ灌流<ol><li>心分離、水浴、水ジャケットを使用して、37℃に暖かいタイロード液の前に。 </li><li> 5％イソフルラン/ 95％O 2と、マウスを麻酔。 </li><li>痛みの反射の消失を確認することにより、麻酔の適切なレベルを確認してください。 </li><li>開胸術を行います。湾曲した5.5 &quot;メイヨーはさみと5.5&quot;ケリーの止血用を使用して、胸を開きます胸部の前面に1cmのカットを作るためにCEPS。 <ol><li>迅速に（30秒以内）胸から心臓を抽出します。 「肺組織をつかむと4.3使用して心膜と一緒に肺、胸腺および心臓をカットするように湾曲アイリス鉗子「アイリスはさみを4を使用してください。直接心をつかむしないでください。 </li><li>酸素（95％O 2/5％CO 2）でそれを洗って、一定温度（36.8±0.4°C）は、タイロード液を変更しました。 128.2のNaCl、4.7のKCl、1.19のNaH 2 PO 4、1.05のMgCl 2、1.3のCaCl 2、20.0のNaHCO 3、および11.1グルコース（pHは7.35±0.05）：以下の溶液組成（mMの中）を使用します。 </li></ol></li><li>同じ溶液に浸しながら、肺、胸腺、および脂肪組織を識別します。慎重に心臓からそれらを分析。湾曲した3「Vannas - テュービンゲンはさみと4.3 &quot;＃5鉗子を使用してください。 </li><li>その後、大動脈を識別し、カスタムメイドの21 Gカニューレの上にカニューレを挿入。 2つの湾曲4.3」＃5Bの力を使用しますpsの。 </li><li>挿管後、同時に灌流;（〜50ml /分の流速で浴中）（〜5ml /分の流速でカニューレを通してこれは大動脈圧に基づいて調整されるべきである、以下参照）、表面かん流します実験全体を通して温め、そして濾過タイロード溶液で心。冠循環の目詰まりを防止するために、インライン11ミクロンのナイロンフィルターを通して灌流液を渡します。 </li><li>灌流ラインに三方活栓ルアーロックを介して接続された圧力変換器（または圧力計）を用いて、大動脈の圧力を監視し、60から80 mmHgの間に維持します。この範囲内で大動脈圧を維持するために必要な場合、灌流速度を調整します。 </li></ol>ランゲンドルフ灌流心臓全体からSANの2光学マッピング<ol><li> 計装 <ol><li>水平に心を置き、Pに直径0.1mmのピンを使用して、チャンバのシリコンコーティングされた底部に心室頂点をピン実験中reventストリーム誘発性運動。12 </li><li>左心房（LA）と僧帽弁（MV）、肺静脈を介して左心室（LV）に小（0.012 &quot;IDのx 0.005&quot;）シリコンチューブを挿入します。響きの結合組織に絹縫合糸（4-0）によりチューブを固定してください。 </li><li>その後方側が（ 図1A、 左パネル）を上にして心を置きます。 </li><li>直径0.1mm minutienピンを用いてチャンバのシリコン底部までのRA心耳（RAA）のエッジをピン。 RAの後面を平坦にし、それはカメラの焦点面に配置されることを可能にするために、そのレベルを調整します。これは、心房の最大表面積から光学的測定を可能にします。 </li><li>絹縫合糸（4-0）による縄優れた（SVC）および（IVC）下大静脈、シリコンコーティングチャンバの底部に縫合糸のもう一方の端を伸ばし、ピン（ 図3Aを参照してください）。縫合糸はブロックしないことを確認してください光学視野。 </li><li> RAAの端にカスタムメイドのペーシング電極を配置します。電極を作製するために、使用したシリコンを0.5mm、電極間距離は、直径0.25mmの銀線を被覆し、ペーシング端1mmの長さのためにシリコンを欠きます。その後、2 ECG 12ミリメートル針（29ゲージ）の右心室と左心室の底部近くモノポーラ電極を配置します。心室の頂点に近いグランドECG電極を配置します。 </li></ol></li><li> 染色 <ol><li>蛍光染料及び電気機械脱共役剤のブレビスタチンの両方が光に敏感であるため、暗い部屋で、以下に記載されているすべての手順を実行します。まず、ジメチルスルホキシド（2.5 mM）の、アリコートそれをストック溶液、1.25 mg / mlでのように（30μLずつ）電位感受性色素RH-237またはジ-4- ANNEPSを準備し、-20℃でアリコートを保存します。 </li><li>温めた（37℃）タイロード溶液1ml色素ストック溶液の5-10μLに希釈した後、冠動脈灌流ラインに注入イン・ラインルアー注入ポートを使用して5-7分間かけて。 </li><li>事前にストック溶液（ジメチルスルホキシド中で2 mg / mlで、6.8 mm）とブレビスタチンを準備し、4℃で保管してください。 </li><li>安定化の20分後、灌流液に温めた0.5mlの（37°C）ブレビスタチンを追加して温めた（37℃）タイロード液1mlでブレビスタチン0.1mlの希薄。次に、インラインルアー注入ポートを使用して5-7分かけて冠動脈灌流ラインに注入。 </li></ol></li></ol>単離された心房準備からSANの3光学マッピング<ol><li> 計装 <ol><li>全心の準備のためのステップ1.1から1.5で説明したように、単離された心房の準備のために、心を分離し、カニューレを挿入。 </li><li>離れた前方側から心室を解剖。 図1A、 右パネルを参照してください、詳細については、＃1を切りました 。 </li><li>三尖弁VAを切断することにより、RAを開きます。TV-SVC軸に沿っLVE（TV）。 図1A、 右パネルを参照してください、詳細については＃2をカット 。 </li><li> RAAを開くには稜終の内側手足をカット。 図1A、 右パネルを参照してください、＃3を切りました 。 </li><li> RAAの右下隅の端に、以前のカット＃3の正中線からのカットを行うことにより、前方心房自由壁を開き、シリコンコーティングチャンバの底部に心房自由壁を平らにし、ピン。 ＃4をカットし 、 図1（a）の白抜き矢印で示す方向を参照してください。心房への損傷を防止するために準備を固定するための心室組織の縁を保持します。 </li><li> LAの付属物（LAA）のMV-上隅に沿ってMVを切断することによりLA同様に、開きました。 </li><li> LAAを開くには、LAAの中央縁近くまで前方心房自由壁を通って、開かれたLAAの真ん中からカット。 </li><li>前方心房自由壁ALOを開きます同じ方向をngの、その後のSylgardコーティングされたチャンバーの底にそれを平らにし、ピン。 </li><li>部分的に心房間中隔組織を削除します。これがフォーカスされていない組織からの光信号の散乱を低減します。したがって、LAおよびRAだけでなく、SANおよび房室接合部（AVJ）の両方がこの準備（ 図1B）でアクセス可能です。 </li><li>心外膜と心内膜面の両方から灌流を可能にするためにシリコンコーティングチャンバの底部から約0.5ミリメートルによる最終準備を持ち上げます。 </li><li>お風呂の灌流のためのSVCおよび〜30ミリリットル/分の近くにある集束されたフローのための〜12ミリリットル/分の一定速度で加温した（37℃）タイロード溶液で準備を表面かん流します。 </li><li>解剖RAAの端にカスタムメイドペーシング銀/塩化銀2電極（直径0.25mm）を配置します。そして、それぞれ、2 ECG 12ミリメートル針（29G）RAAおよびLAA近い電極（単極）を配置。地面ECG当選者を配置AVJの近くに乗りました。 </li></ol></li><li> 染色 <ol><li>電位感受性色素（RH-237またはジ-4- ANNEPS）の直接適用を実行します。このために、暖めた（37℃）タイロード溶液1mmの色素ストック溶液を1-2μLに希釈し、ゆっくりと、1mmのピペットを使用して製剤の表面上に希釈された色素を放出します。 </li><li>また、全体の心の準備（ステップ2.2.1-2.2.2）のために上記と同様のランゲンドルフ灌流心臓の冠状動脈の灌流を介して電圧感受性色素を持つ心房染色を行います。 <ol><li>記載されているように冠動脈灌流染色した後、心房準備を隔離します。十分な蛍光レベルに到達するために必要なときに追加の表面染色を実行します。クイック心房分離は、色素を漂白しないと染色の品質には影響しません。 </li></ol></li><li>電気機械脱共役剤、ブレビスタチンと準備を固定化。 BLE 3-5μLを希釈温めておいた（37℃）タイロード溶液中の原液bbistatin、ゆっくり準備と周囲の溶液の表面に希釈したブレビスタチンをリリース。ブレビスタチンので13,14が準備し、染色時の光に長時間露光を避けるため、光に敏感であることが示されています。 </li><li>収縮を抑制するために必要な量の追加のブレビスタチンのアプリケーションを実行します。通常、3つの追加アプリケーション（各アプリケーション当たり3~5μL）まで正確にSANおよび心房活性化を再構成するために十分なモーションアーチファクトを抑制するために使用することができます。 </li><li>灌流液中で温めたブレビスタチンの追加の0.5ミリリットルを追加します。この手順の間、心房組織を染色する色素/ブレビスタチンを可能にするために30秒間灌流を一時停止します。 </li></ol></li></ol> 4.光学マッピングの設定します注：光学マッピングシステムの詳細な説明は他の場所に提供されている12。 <ol><li> TEMでカメラを使用poral 2,000フレーム/秒以上の解像度、及び100ミクロン/ピクセル以上の空間分解能に到達する集光レンズのシリーズ。これは、SAN活性化および結節内伝播を再構成するために必要とされます。 </li><li>振動溶液からモーションアーチファクトを低減するために、心臓/孤立性心房準備の上、溶液の表面に小さなカバーガラスを固定します。 </li><li>定電流、低ノイズのハロゲンランプが提供する励起光（520±45 nmの波長）を使用します。柔軟な二股ライトガイドを使用して準備にフィルタされた光ビームを向けます。 </li><li>ロングパスフィルタ（&gt; 715 nm）を発する蛍光をフィルタリングします。収集し、デジタル化し、カメラメーカーが提供するソフトウェアを使用しているコンピュータ上で、取得した蛍光シグナルを保存します。 </li></ol> 5.データ処理注記：光学的マッピングデータを収集し、異なる時点での蛍光強度の行列の列として格納されます。各画素represe特定の時点での組織標本上の特定の位置から採取した蛍光強度の測定をNTS。バックグラウンド蛍光は、自動的に、膜電位の変化によって生成され、微小電極システムによって測定された活動電位（APS）に対応して倒立蛍光変化のより良好な視覚化を可能にするために、画素毎に除去されます。画像分割、空間フィルタリング、時間フィルタリング、ベースラインドリフト除去を含む光学撮像データを処理における様々なステップの詳細、集束レビューに設けられている。15 <ol><li>手動で、または組織の境界を決定するための特別なアルゴリズム（例えば、閾値化、エッジ検出）15を用いて、1（含まれる画素）と0（除外画素）のバイナリマスクを作成し、シーケンス内の各フレームにマスクを適用します。このプロセスは、さらなる処理のために関心のある領域を強調した二値画像を作成します。 </li><li> OPTの信号対雑音比を改善するためにiCalのデータは、関心のある選択された領域内の信号をフィルタリングします。このため、空間的な（ すなわち 、所望の畳み込みビンまたはカーネルによって定義されるように、隣接する蛍光画素の平均値、例えば3×3または、ノイズの多いデータ、5×5の場合）、および/または時間フィルタリング（例えば、バターワース、チェビシェフ型を使用1、チェビシェフ型2、楕円等 ）（ 図2）。データを解釈する際に念頭に置いて、ろ過によって誘発されるアーティファクトの可能性をキープ。詳細については、レビューを参照してください。15 </li><li>必要なときに（元の信号にハイパスフィルタまたは多項式フィッティングを使用して）、光学録音でベースラインのドリフトを削除してから1に0（最小蛍光）から各画素信号を正規化（最大蛍光）。 </li><li>単一のAP伝播の全画素の活性化時間を含む時間ウィンドウを選択します。 Fはfluorescenで最大アップストローク誘導体（DF / dtの最大の時間として、各画素に、その起動時間を割り当てCE強度）。全画素活性化時間を使用して、等時間活性化マップを再構築します。各アイソクロンは、このように同時に作動ピクセルが表示されます。 </li><li> AP持続時間（APD）の分布マップを再構築するために、画素ごとに（例えば、再分極の80％で、APD 80）再分極の特定のレベルでの活性化時間と時間の間の持続時間を計算します。全ての画素APDの値を使用して、APD分布等時マップを再構築します。各アイソクロンは、このように同じAPDを持つピクセルが表示されます。 </li><li> APの伝播の伝導速度を計算するために、5.4で算出起動時間データに製剤の表面に合います。このために、多項式表面フィッティングまたはローカルカーネルの表面平滑化を使用し、フィット表面の勾配からローカル伝導速度ベクトルを算出します。 </li><li>再分極マップを作成するには、各ピクセルにその再分極時間を割り当て、最大の第2導関数として定義される（D 2 F / dtは2）OAP、または90％の再分極の時間の終わりに光信号の。 </li></ol>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+blebbistatin+for+cardiac+optical+mapping+and+other+imaging+applications.">Properties of blebbistatin for cardiac optical mapping and other imaging applications.</a> Pflugers Arch. 464 (5), 503-512 (2012).</li><li>Laughner, J. I., Ng, F. S., Sulkin, M. S., Arthur, R. M., 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=Processing+and+analysis+of+cardiac+optical+mapping+data+obtained+with+potentiometric+dyes.">Processing and analysis of cardiac optical mapping data obtained with potentiometric dyes.</a> Am J Physiol Heart Circ Physiol. 303 (7), H753-H765 (2012).</li><li>Liu, J., Dobrzynski, H., Yanni, J., Boyett, M. R., Lei, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organisation+of+the+mouse+sinoatrial+node:+structure+and+expression+of+HCN+channels.">Organisation of the mouse sinoatrial node: structure and expression of HCN channels.</a> Cardiovasc Res. 73 (4), 729-738 (2007).</li><li>Verheijck, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+features+of+the+mouse+sinoatrial+node+in+relation+to+connexin+distribution.">Electrophysiological features of the mouse sinoatrial node in relation to connexin distribution.</a> Cardiovasc Res. 52 (1), 40-50 (2001).</li><li>Krishnaswamy, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+parasympathetic+nervous+system+regulation+of+the+sinoatrial+node+in+Akita+diabetic+mice.">Altered parasympathetic nervous system regulation of the sinoatrial node in Akita diabetic mice.</a> J Mol Cell Cardiol. 82, 125-135 (2015).</li><li>Nygren, A., Lomax, A. E., Giles, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+action+potential+durations+in+isolated+mouse+left+and+right+atria+recorded+using+voltage-sensitive+dye+mapping.">Heterogeneity of action potential durations in isolated mouse left and right atria recorded using voltage-sensitive dye mapping.</a> Am J Physiol Heart Circ Physiol. 287 (6), H2634-H2643 (2004).</li><li>Efimov, I. R., Fedorov, V. V., Joung, B., Lin, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+cardiac+pacemaker+circuits:+methodological+puzzles+of+the+sinoatrial+node+optical+mapping.">Mapping cardiac pacemaker circuits: methodological puzzles of the sinoatrial node optical mapping.</a> Circ Res. 106 (2), 255-271 (2010).</li><li>Aschar-Sobbi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+atrial+arrhythmia+susceptibility+induced+by+intense+endurance+exercise+in+mice+requires+TNFalpha.">Increased atrial arrhythmia susceptibility induced by intense endurance exercise in mice requires TNFalpha.</a> Nat Commun. 6, 6018 (2015).</li><li>Hansen, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrial+fibrillation+driven+by+micro-anatomic+intramural+re-entry+revealed+by+simultaneous+sub-epicardial+and+sub-endocardial+optical+mapping+in+explanted+human+hearts.">Atrial fibrillation driven by micro-anatomic intramural re-entry revealed by simultaneous sub-epicardial and sub-endocardial optical mapping in explanted human hearts.</a> Eur Heart J. 36 (35), 2390-2401 (2015).</li><li>Lang, D., Petrov, V., Lou, Q., Osipov, G., 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=Spatiotemporal+control+of+heart+rate+in+a+rabbit+heart.">Spatiotemporal control of heart rate in a rabbit heart.</a> J Electrocardiol. 44 (6), 626-634 (2011).</li><li>Kanlop, N., Sakai, T. <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+study+of+blebbistatin-induced+chaotic+electrical+activities+in+isolated+rat+atrium+preparations.">Optical mapping study of blebbistatin-induced chaotic electrical activities in isolated rat atrium preparations.</a> J Physiol Sci. 60 (2), 109-117 (2010).</li><li>Glukhov, A. V., Flagg, T. P., Fedorov, V. V., Efimov, I. R., Nichols, C. G. <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. 48 (1), 152-160 (2010).</li><li>Wu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+sinoatrial+node+function+and+intra-atrial+conduction+in+murine+gain-of-function+Scn5a+/DeltaKPQ+hearts+suggest+an+overlap+syndrome.">Altered sinoatrial node function and intra-atrial conduction in murine gain-of-function Scn5a+/DeltaKPQ hearts suggest an overlap syndrome.</a> Am J Physiol Heart Circ Physiol. 302 (7), H1510-H1523 (2012).</li></ol>

No conflicts of interest are declared.

ランゲンドルフ灌流心臓全体で1）無傷のSAN、および2）SANは、単離された中で、心房の準備を開いた：ここでは、マウスのSAN製剤の2種類を発表しました。製剤のこれらの2つのタイプは、異なる実験的な目的を果たします。ランゲンドルフ灌流心臓全体の製造においては、無傷の心房構造は、心房細動と同様に再入頻脈性不整脈の間にSANと心房の間の相互作用などの複雑な心房性不整脈を研究...

Novel scientific breakthroughs that lead to leaps in the understanding of human physiology are often preceded by technological advances. Fluorescent optical mapping, for example, enables investigation of multiple physiological parameters in both cells and tissues.1,2 It significantly improved our understanding of how the anatomical structure is associated with electrophysiological functions and dysfunctions. In the heart, the natural pacemaker and conduction systems such as the sinoatrial node (SAN) and the atrioventricular junction consist of nodal myocytes that are insulated by the surrounding atrial myocytes.3-5 Such organization creates complex three-dimensional structures with specialized electrical properties. Both structural and functional remodeling of these pacemaker structures has been recognized to form significant electrophysiological heterogeneities.6,7 Understanding the mechanism underlying how such heterogeneities result in SAN dysfunctions and atrial arrhythmogenesis will dramatically benefit the clinical treatment of these diseases. It requires a technique to visualize the propagation of electrical signals at the tissue level, such as optical mapping. 
Recently, accumulating evidence has proven the advance of optical mapping in studies of atrial electrophysiology and pathology.2 However, novel and rigorous studies are dependent on the accurate interpretation of experimental data, whose validity and stability rely on careful experiment protocols. Genetically modified mice are extensively used for research as animal models of human diseases including sick sinus syndrome, pacemaker abnormalities and atrial arrhythmias.6,8-11 Thus, a combination of fluorescent optical mapping with transgenic mouse models provides a powerful tool to study cardiac electrical abnormalities associated with various pathologies. In this paper, we present a protocol for high-resolution optical mapping of the mouse SAN and atrium. Specifically, we discuss and compare different dye loading approaches, time effects on dye bleaching and heart rate stability during the experiment.

<table><tbody><tr><td>water jacket</td><td>Radnoti</td><td>1660 Series Tissue Bath for Large Organ or Single Cell Isolation Procedures</td><td></td></tr><tr><td>water bath / circulator</td><td>Fisher Scientific</td><td>1016S</td><td></td></tr><tr><td>pressure amplifier</td><td>AD Instruments</td><td>MLT0670</td><td></td></tr><tr><td>EMD Millipore Nylon Net Filters</td><td>Fisher Scientific</td><td>NY1102500</td><td></td></tr><tr><td>Pressure transducer</td><td>AD Instruments</td><td>MLT0670</td><td></td></tr><tr><td>Stainless Steel Minutien Pins - 0.1 mm Diameter</td><td>Fine Science Tools</td><td>26002-10&amp;nbsp;</td><td></td></tr><tr><td>Perfusion pump</td><td>World Precision Instruments</td><td>PERIPRO-4LS</td><td></td></tr><tr><td>Superfusion pump</td><td>World Precision Instruments</td><td>PERIPRO-4HS</td><td></td></tr><tr><td>Vannas Tubingen scissors&amp;nbsp;</td><td>World Precision Instruments</td><td>503379</td><td></td></tr><tr><td>Dumont forceps</td><td>World Precision Instruments</td><td>501201, 500085</td><td></td></tr><tr><td>Mayo scissors</td><td>World Precision Instruments</td><td>501750</td><td></td></tr><tr><td>Kelly hemostatic forceps</td><td>World Precision Instruments</td><td>501241</td><td></td></tr><tr><td>Iris forceps</td><td>World Precision Instruments</td><td>15917</td><td></td></tr><tr><td>Iris scissors</td><td>World Precision Instruments</td><td>501263</td><td></td></tr><tr><td>ECG 12 mm needle (29-gauge) electrodes (monopolar)&amp;nbsp;</td><td>AD Instruments</td><td>MLA1203</td><td></td></tr><tr><td>in-line Luer injection port</td><td>Ibidi</td><td>10820</td><td></td></tr><tr><td>Ultima-L CMOS camera&amp;nbsp;</td><td>SciMedia</td><td>MiCAM-05&amp;nbsp;</td><td></td></tr><tr><td>halogen lamp</td><td>Moritex USA Inc</td><td>MHAB-150W</td><td></td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>S271-1</td><td></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></td></tr><tr><td>KCl</td><td>Fisher Scientific</td><td>S217-500</td><td></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></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></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher Scientific</td><td>S233-3</td><td></td></tr><tr><td>D-Glucose</td><td>Fisher Scientific</td><td>D16-1</td><td></td></tr><tr><td>Blebbistatin</td><td>Tocris Bioscience</td><td>1760</td><td></td></tr><tr><td>RH237</td><td>ThermoFisher Scientific</td><td>S1109</td><td></td></tr><tr><td>Dimethyl sulphoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr></tbody></table>

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.

ランゲンドルフ灌流心臓から無傷SANの光学マッピング 自発的な洞調律のために再構成されたRA活性化等高線の典型的な例は、ランゲンドルフ灌流マウス心臓については、 図3に示されています。早期活性化点がSANを解剖学的に定義されているSVC周辺intercaval領域内に位置している。1.0および0.5ミリ秒のサンプリングレートで取得3,16</sup...

Watch this Scientific Journal Video about High-resolution Optical Mapping of the Mouse Sino-atrial Node at JoVE.com

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

ここでは、高い空間分解能と時間分解能で、マウス右心房、特に洞房結節から電気活動の光学マッピングするためのプロトコルを提示します。

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 ...

high-resolution-optical-mapping-of-the-mouse-sino-atrial-node

Research

JoVE Journal

Biology

15.6K ビュー.  University of Wisconsin-Madison School of Medicine and Public Health. ここでは、高い空間分解能と時間分解能で、マウス右心房、特に洞房結節から電気活動の光学マッピングするためのプロトコルを提示します。 

ビデオ: High-resolution Optical Mapping of the Mouse Sino-atrial Node

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

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 ...

包括的な心臓イメージングのためのライトシート顕微鏡検査と組織透明化

Light-Sheet Imaging to Reveal Cardiac Structure in Rodent Hearts

Light-sheet microscopy (LSM) plays a pivotal role in comprehending the intricate three-dimensional (3D) structure of the heart, providing crucial insights into fundamental cardiac physiology and pathologic responses. We hereby delve into the development and implementation of the LSM technique to elucidate the micro-architecture of the heart in mouse models. The methodology integrates a customized LSM system with tissue clearing techniques, mitigating light scattering within cardiac tissues for volumetric imaging. The combination of conventional LSM with image stitching and multiview deconvolution approaches allows for the capture of the entire heart. To address the inherent trade-off between axial resolution and field of view (FOV), we further introduce an axially swept light-sheet microscopy (ASLM) method to minimize out-of-focus light and uniformly illuminate the heart across the propagation direction. In the meanwhile, tissue clearing methods such as iDISCO enhance light penetration, facilitating the visualization of deep structures and ensuring a comprehensive examination of the myocardium throughout the entire heart. The combination of the proposed LSM and tissue clearing methods presents a promising platform for researchers in resolving cardiac structures in rodent hearts, holding great potential for the understanding of cardiac morphogenesis and remodeling.

著者スポットライト:げっ歯類の心臓の心筋構造を調査するためのカスタマイズされたライトシートイメージング

The protocol utilizes advanced light-sheet microscopy along with adapted tissue clearing methods to investigate intricate cardiac structures in rodent hearts, holding great potential for the understanding of cardiac morphogenesis and remodeling.

げっ歯類の心臓の構造を明らかにするためのライトシートイメージング

Light-sheet microscopy (LSM) plays a pivotal role in comprehending the intricate three-dimensional (3D) structure of the heart, providing crucial insights ...

Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse

The electrical signal physiologically generated by pacemaker cells in the sinoatrial node (SAN) is conducted through the conduction system, which includes the atrioventricular node (AVN), to allow excitation and contraction of the whole heart. Any dysfunction of either SAN or AVN results in arrhythmias, indicating their fundamental role in electrophysiology and arrhythmogenesis. Mouse models are widely used in arrhythmia research, but the specific investigation of SAN and AVN remains challenging.
The SAN is located at the junction of the crista terminalis with the superior vena cava and AVN is located at the apex of the triangle of Koch, formed by the orifice of the coronary sinus, the tricuspid annulus, and the tendon of Todaro. However, due to the small size, visualization by conventional histology remains challenging and it does not allow the study of SAN and AVN within their 3D environment.
Here we describe a whole-mount immunofluorescence approach that allows the local visualization of labelled mouse SAN and AVN. Whole-mount immunofluorescence staining is intended for smaller sections of tissue without the need for manual sectioning. To this purpose, the mouse heart is dissected, with unwanted tissue removed, followed by fixation, permeabilization and blocking. Cells of the conduction system within SAN and AVN are then stained with an anti-HCN4 antibody. Confocal laser scanning microscopy and image processing allow differentiation between nodal cells and working cardiomyocytes, and to clearly localize SAN and AVN. Furthermore, additional antibodies can be combined to label other cell types as well, such as nerve fibers.
Compared to conventional immunohistology, whole-mount immunofluorescence staining preserves the anatomical integrity of the cardiac conduction system, thus allowing the investigation of AVN; especially so into their anatomy and interactions with the surrounding working myocardium and non-myocyte cells.

We provide a step-by-step protocol for whole-mount immunofluorescence staining of the sinoatrial node (SAN) and atrioventricular node (AVN) in murine hearts.

マウスの洞房結節と房室結節のホールマウント免疫蛍光染色、共焦点イメージング、3D再構成

The electrical signal physiologically generated by pacemaker cells in the sinoatrial node (SAN) is conducted through the conduction system, which includes ...

Microelectrode Array Recording of Sinoatrial Node Firing Rate to Identify Intrinsic Cardiac Pacemaking Defects in Mice

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or bradycardia. Reliable identification of cardiac pacemaking defects requires the measurement of intrinsic heart rates by largely preventing the influence of the autonomic nervous system, which can mask rate deficits. Traditional methods to analyze intrinsic cardiac pacemaker function include drug-induced autonomic blockade to measure in vivo heart rates, isolated heart recordings to measure intrinsic heart rates, and sinoatrial strip or single-cell patch-clamp recordings of sinoatrial pacemaker cells to measure spontaneous action potential firing rates. However, these more traditional techniques can be technically challenging and difficult to perform. Here, we present a new methodology to measure intrinsic cardiac firing rate by performing microelectrode array (MEA) recordings of whole-mount sinoatrial node preparations from mice. MEAs are composed of multiple microelectrodes arranged in a grid-like pattern for recording in vitro extracellular field potentials. The method described herein has the combined advantage of being relatively faster, simpler, and more precise than previous approaches for recording intrinsic heart rates, while also allowing easy pharmacological interrogation.

This protocol aims to describe a new methodology to measure intrinsic cardiac firing rate using microelectrode array recording of the whole sinoatrial node tissue to identify pacemaking defects in mice. Pharmacological agents can also be introduced in this method to study their effects on intrinsic pacemaking.

マウスにおける心内ペース作りの欠損を同定するための、中房ノードの発火率のマイクロ電極アレイ記録

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or ...

マウス洞房結節の高分解能光学マッピング

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

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

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

ランゲンドルフ灌流ラット心臓の光学的マッピング

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

げっ歯類の心臓の構造を明らかにするためのライトシートイメージング

げっ歯類の心臓の構造を明らかにするためのライトシートイメージング

げっ歯類の心臓の構造を明らかにするためのライトシートイメージング

マウスの洞房結節と房室結節のホールマウント免疫蛍光染色、共焦点イメージング、3D再構成

マウスにおける心内ペース作りの欠損を同定するための、中房ノードの発火率のマイクロ電極アレイ記録