この研究は、ドイツ研究財団(DFG;血管医学の臨床医科学者プログラム(PRIME)、MA 2186 / 14-1からP.トムシッツおよびD.シュットラーへ。VO1568/3-1、IRTG1816、およびSFB1002プロジェクトA13からN.フォークト)、ドイツの卓越性戦略の下でのドイツ研究財団(EXC 2067/1-390729940からN.フォークト)、ドイツ心臓血管研究センター(DZHK; 81X2600255からS.クラウスおよびN.フォークト; 81Z0600206からS.ケーブ)、コロナ財団(S199/10079/2019からS.クラウス)、心血管疾患に関するERA-NET(ERA-CVD、01KL1910からS.クラウス)、 ハインリッヒ・アンド・ロッテ・ミュールフェンツル財団(S.クラウスへ)とエルゼ・クレーナー・フレゼニウス財団(EKFS 2016_A20 N.フォークトへ)。資金提供者は原稿の準備に何の役割も果たしていませんでした。

すべての動物の手順は、ニーダーザクセン州動物審査委員会(LAVES、AZ-18 / 2900)によって承認され、動物福祉に関するすべての制度的、国内的、および国際的なガイドラインに従って実施されました。
1. 事前手配
<ol><li>1 Lの10x灌流バッファー(表1)、500 mLの1x灌流バッファー(表2)、50 mLの消化バッファー(表3)、10 mLのストップバッファー(表4)、1 Lのタイロード溶液(表5)、10 mLの各カルシウムステップ溶液(示されているようにグルコースおよびそれぞれの量のカルシウムを含むタイロード溶液)を準備します。 提供されたレシピに従って、1 Lの4-AP溶液(表5)、100 mLのピペット溶液(表6)および5 mLのプルロン酸。 注:浴溶液(タイロードおよび4-AP溶液)は、事前に調製し(グルコースなし)、+ 4°Cで保存することができ、グルコースは実験日に添加される。ピペット溶液は-20°Cで保存でき、実験日にカルシウム指示薬を添加し、溶液をさらに使用するまで氷上で保存します。10x灌流バッファーは室温で保存でき、1x灌流バッファー、消化バッファー、および停止バッファーは実験日に新たに調製する必要があります。</li>
	<li>ウォーターバスとローラーポンプをオンにします。</li>
	<li>ランゲンドルフ装置に灌流バッファーをプレフィルします。空気がないことを確認してください。</li>
	<li>大動脈カニューレを解剖顕微鏡で固定して調製し、灌流バッファーときれいな空気で満たされた1 mLシリンジでカニューレをすすぎます。 注:灌流システム内の空気は冠状動脈灌流に直接影響し、消化効果に影響を与えるため、避けることが重要です。エアトラップを安全に回避するために、必要に応じてバブルトラップをセットアップに追加できます。</li>
	<li>臓器採取と顕微鏡検査に十分な灌流バッファーを使用してペトリ皿を準備します(バッファーは臓器全体をしっかりと覆う必要があり、使用するペトリ皿のサイズに応じて数ミリリットルで十分です)。</li>
	<li>組織解離および顕微鏡検査のための消化バッファーで3つのペトリ皿を準備し、緩衝液はそれぞれのペトリ皿内の顕微鏡下で解剖のために器官を覆うべきであり、量は使用されるペトリ皿のサイズに依存する。解離には、心室組織に3 mL、心房組織に1.5 mLを使用します。</li>
</ol>2.臓器摘出
<ol><li>27 Gカニューレを備えた1 mLシリンジを使用して、0.1 mLのヘパリン(1,000 U / mL)をマウスに注射し、5〜10分待ちます。</li>
	<li>マウスを、約500 μLのイソフルランに浸した小さな組織と一緒に誘導チャンバーに入れます。動物は組織と接触してはいけません。それを避けるために、プラスチック製の生検埋め込みカセットを使用して組織を覆うことができます。動物が完全に麻酔されたら、つま先のつまみ反射をチェックし、存在しなくなったらすぐに、頸部脱臼によってマウスをすぐに安楽死させます。</li>
	<li>マウスを背中のプラットフォーム(ペーパータオルで覆われた発泡スチロールなど)に置き、カニューレで足を固定して所定の位置に保持します。</li>
	<li>胸部と腹部の一部を覆っている毛皮と皮膚をジュグルムから結合に向かってはっきりと切り込み、ハサミを使用して臓器構造を傷つけることなく剣状突起の真下腹部を開きます。外科用鉗子で胸骨を持ち上げ、肋骨の端に沿ってハサミで横隔膜を切断し、次に腋窩の内側線で肋骨を切断し、胸郭を取り外して心臓を露出させます。</li>
	<li>鈍い鉗子を使用して心膜を慎重に取り外し、下から鈍い鉗子で持ち上げ、ハサミを使用して1回の切断で大きな血管を切断することにより、心臓をすばやく取り除きます。</li>
	<li>心臓を室温灌流バッファーに入れ、顕微鏡下で鈍い端の針で大動脈をできるだけ早くカニューレ挿入します。 注意: 臓器に付着している肺組織や脂肪組織を、時間をあまり失うことなく取り除きます。カニューレ挿入中は、緩衝液が冠状動脈に入るのを防ぐことによって結果を損なうため、針の端が大動脈弁を通って伸びないようにしてください。</li>
	<li>縫合シルクで心臓を針にしっかりと結び、注射器から外します。 注:心臓の取得(大きな血管が切断された瞬間)から大動脈を針に縫合するまでの全手順は、できるだけ短い時間で済みます。心臓の除去から灌流の開始まで90〜180秒以上かからないことをお勧めします。</li>
</ol>3.酵素消化
<ol><li>大動脈カニューレ挿入後、すぐにカニューレをランゲンドルフ装置に接続し、システムに空気が入らないようにします。 注:ランゲンドルフ装置の下部に灌流バッファーのドロップをぶら下げ、システムに空気が入らないようにするために、針の上部に灌流バッファーのドロップを置くと役立ちます。</li>
	<li>心臓に灌流バッファーを1分間、温度37°C、灌流速度4 mL/minで灌流します。 注:灌流針の先端の温度を37°Cにするには、ウォーターバスの温度を約40°Cよりわずかに高く設定する必要があります。 これは、灌流先端の温度を測定することによって定期的にテストする必要があります。</li>
	<li>灌流を消化バッファーに切り替え、正確に37°Cの温度と正確に4mL/minの灌流速度で正確に9分間灌流します。</li>
	<li>消化された心臓を完全に覆うのに十分な消化バッファーを備えたペトリ皿に移します。次に、顕微鏡で心房と心室を慎重に解剖します。</li>
	<li>心房を1.5 mLの消化バッファーを含むペトリ皿に移し、心室を3 mLの消化バッファーを含む別のペトリ皿に移します。</li>
	<li>心房郭清<ol><li>慎重に、しかし時間を失うことなく、鈍い鉗子を使用して心房を小さな断片に引き離します。</li>
		<li>チップの開口部を広げるために以前に切断された1,000 μLのピペットチップを使用して慎重に上下にピペッティングして、組織を溶解します。</li>
		<li>溶液を15 mLの遠沈管に移し、チューブの側面を注意深くピペッティングして等量のストップバッファー(1.5 mL)を加えて反応を終了します。</li>
		<li>3 mLの細胞/組織溶液すべてを200 μmのナイロンメッシュに注意深く通し、完全に消化されていない残りの大きな組織片を取り除きます。 注:消化が成功すると、固体の塊はほとんど残りません。</li>
	</ol></li>
	<li>心室解離<ol><li>解剖ハサミとピペットを上下に使用して、心室組織をすばやく細かく切り刻んで溶解します。上下にピペッティングするために別の1,000μLピペットチップを使用し、開口部を広げるために短くしてもよい。</li>
		<li>細胞/組織溶液を15 mLの遠沈管に移し、チューブの側面で慎重にピペッティングして等量のストップバッファー(3 mL)を加えて反応を終了します。</li>
		<li>6 mLの細胞/組織溶液すべてを200 μmのナイロンメッシュに注意深く通し、完全に消化されていない大きな組織片を取り除きます。 注:消化が成功すると、固体の塊はほとんど残りません。</li>
	</ol></li>
	<li>両方のチューブ(心房および心室細胞懸濁液)を室温で6分間ベンチに置いておいて落ち着かせます。</li>
	<li>両方の15 mLチューブを5 x g で2分間遠心分離します。</li>
</ol>4.カルシウムの再導入
注:次の手順は、心房細胞と心室細胞の両方で同じであり(特に言及されていない限り)、室温で実行されます。
<ol><li>プラスチック製のパスツールピペットを使用して上清を廃棄し、ペレットを10 mLのカルシウムフリータイロード溶液に注意深く再懸濁します。</li>
	<li>沈降のために細胞を8分間放置します。</li>
	<li>5 x g で1分間遠心分離する(心房細胞のみ、沈降は心室細胞には十分である)。</li>
	<li>上清を廃棄し、カルシウム濃度100 μMのタイロード溶液10 mLにペレットを注意深く再懸濁します。</li>
	<li>沈降のために細胞を8分間放置します。</li>
	<li>5 x g で1分間遠心分離する(心房細胞のみ、沈降は心室細胞には十分である)。</li>
	<li>上清を廃棄し、カルシウム濃度400 μMの10 mLのタイロード溶液にペレットを注意深く再懸濁します。</li>
	<li>沈降のために細胞を8分間放置します。</li>
	<li>5 x g で1分間遠心分離する(心房細胞のみ、沈降は心室細胞には十分である)。</li>
	<li>上清を廃棄し、ペレットを1 mL(心房)/ 5 mL(心室)のタイロード溶液に1 mMカルシウム濃度で注意深く再懸濁します。.</li>
</ol>5.蛍光カルシウム指示薬Fluo-3 AMによる筋細胞の負荷
注意: 蛍光カルシウムインジケーターの光感度のため、次の手順は光から保護して実行する必要があります(たとえば、チューブをアルミホイルで覆うなど)。
<ol><li>無水DMSO中の20%プルロニックF-127を50 μgのFluo-3AMに添加して、Fluo-3 AMストック溶液を調製します(光から保護された-20°Cで保存できます)。</li>
	<li>10 μLのFluo-3 AMストック溶液を1 mLの細胞懸濁液に加え、光から保護された室温で10分間インキュベートします。</li>
	<li>5 x g で1分間遠心分離します。</li>
	<li>プラスチック製のパスツールピペットを使用して上清を廃棄し、ペレットを妥当な量の浴溶液に再懸濁して、良好な使用濃度(細胞密度に応じて1〜5 mLの浴溶液)を得ます。</li>
	<li>実験を開始する前に、脱エステル化のために30分間放置してください。</li>
</ol>6.前述のパッチクランプとエピ蛍光Ca2+過渡測定の同時測定16
注:パッチクランプ測定はこの記事のトピックではなく、関心のある読者は、この方法の詳細な説明を提供する主要な出版物を参照することができます17、18、19、20、21、22。それにもかかわらず、全体的な理解を深めるために、電流誘発カルシウム過渡現象とともにL型カルシウム電流を測定するためのプロトコルに関する要約が提供されています。
<ol><li>筋細胞を細胞室に移し、37°Cの浴液で過溶します。</li>
	<li>表5に示すように、4-アミノピリジンと塩化バリウムを浴溶液に添加することにより、カリウム電流を遮断します。</li>
	<li>ホウケイ酸微小電極の先端抵抗がピペット溶液で満たされた2〜5MΩであることを確認してください(表6)。</li>
	<li>電気信号と落射蛍光の両方を同時に記録できるように測定を設定します。電圧クランプモードを使用して、セルを-80mVに保持し、600msのランプパルスを-40mVに保持して高速Na+電流を非アクティブ化し、続いて0.5Hzで+10mVまで100msのテストパルスを使用してL型Ca2+電流を測定します(図2)。</li>
	<li>488 nmで励起を使用し、<520 nmで発光して検出し、[Ca 2+]I に変換すると仮定します <img alt="Equation 1" src="/files/ftp_upload/61964/61964equ01.jpg"> ここで、kd = Fluo-3の解離定数(864 nM)、F = Fluo-3蛍光;Fmax=Ca2+-飽和蛍光は各実験19の終了時に得られた。</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel,+rapid+and+reversible+method+to+measure+Ca+buffering+and+time-course+of+total+sarcoplasmic+reticulum+Ca+content+in+cardiac+ventricular+myocytes.">A novel, rapid and reversible method to measure Ca buffering and time-course of total sarcoplasmic reticulum Ca content in cardiac ventricular myocytes.</a> Pflugers Archiv: European Journal of Physiology. 437 (3), 501-503 (1999).</li><li>Hamill, O. P., Marty, A., Neher, E., Sakmann, B., Sigworth, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+patch-clamp+techniques+for+high-resolution+current+recording+from+cells+and+cell-free+membrane+patches.">Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.</a> European Journal of Physiology. 391 (2), 85-100 (1981).</li><li>Voigt, N., 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=Enhanced+sarcoplasmic+reticulum+Ca2++leak+and+increased+Na+-Ca2++exchanger+function+underlie+delayed+afterdepolarizations+in+patients+with+chronic+atrial+fibrillation.">Enhanced sarcoplasmic reticulum Ca2+ leak and increased Na+-Ca2+ exchanger function underlie delayed afterdepolarizations in patients with chronic atrial fibrillation.</a> Circulation. 125 (17), 2059-2070 (2012).</li><li>Fakuade, F. 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=Altered+atrial+cytosolic+calcium+handling+contributes+to+the+development+of+postoperative+atrial+fibrillation.">Altered atrial cytosolic calcium handling contributes to the development of postoperative atrial fibrillation.</a> Cardiovascular Research. , (2020).</li><li>Chen, W., Frangogiannis, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+inflammatory+and+fibrogenic+pathways+in+heart+failure+associated+with+aging.">The role of inflammatory and fibrogenic pathways in heart failure associated with aging.</a> Heart Failure Reviews. 15 (5), 415-422 (2010).</li><li>Pla&#269;ki&#263;, J., Kocksk&#228;mper, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+atrial+and+ventricular+cardiomyocytes+for+in+vitro+studies.">Isolation of atrial and ventricular cardiomyocytes for in vitro studies.</a> Methods in Molecular Biology. 1816, 39-54 (2018).</li><li>D&#237;az, M. E., Trafford, A. W., Eisner, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+exogenous+calcium+buffers+on+the+systolic+calcium+transient+in+rat+ventricular+myocytes.">The effects of exogenous calcium buffers on the systolic calcium transient in rat ventricular myocytes.</a> Biophysical Journal. 80 (4), 1915-1925 (2001).</li><li>Zimmerman, A. N., H&#252;lsmann, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paradoxical+influence+of+calcium+ions+on+the+permeability+of+the+cell+membranes+of+the+isolated+rat+heart.">Paradoxical influence of calcium ions on the permeability of the cell membranes of the isolated rat heart.</a> Nature. 211 (5049), 646-647 (1966).</li><li>Chen, X., O'Connell, T. D., Xiang, Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=With+or+without+Langendorff:+A+new+method+for+adult+myocyte+isolation+to+be+tested+with+time.">With or without Langendorff: A new method for adult myocyte isolation to be tested with time.</a> Circulation Research. 119 (8), 888-890 (2016).</li><li>Kappadan, V., 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=High-resolution+optical+measurement+of+cardiac+restitution,+contraction,+and+fibrillation+dynamics+in+beating+vs.+blebbistatin-uncoupled+isolated+rabbit+hearts.">High-resolution optical measurement of cardiac restitution, contraction, and fibrillation dynamics in beating vs. blebbistatin-uncoupled isolated rabbit hearts.</a> Frontiers in Physiology. 11, 464 (2020).</li><li>Brack, K. E., Narang, R., Winter, J., Ng, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanical+uncoupler+blebbistatin+is+associated+with+significant+electrophysiological+effects+in+the+isolated+rabbit+heart.">The mechanical uncoupler blebbistatin is associated with significant electrophysiological effects in the isolated rabbit heart.</a> Experiment in Physiology. 98 (5), 1009-1027 (2013).</li><li>Seibertz, F., Reynolds, M., Voigt, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+optical+action+potential+measurement+in+human+induced+pluripotent+stem+cell-derived+cardiomyocytes.">Single-cell optical action potential measurement in human induced pluripotent stem cell-derived cardiomyocytes.</a> Journal of Visual Experiment. , e61890 (2020).</li></ol>

この記事では、カルシウム一過性記録を同時に行うパッチクランプ研究のために、同じマウスから高品質の心房筋細胞と心室筋細胞を取得する簡単で機能的な方法を提供します。得られたデータの品質は、細胞単離の品質に大きく依存します。上述のように、マウス心筋細胞を単離する多くの方法が以前に記載されている9、10、11...

心不整脈は一般的であり、世界中の何百万人もの人々に影響を与えるため、現在の主要な医療課題の1つです。不整脈は高い罹患率と死亡率1,2に関連しており、心臓突然死の大部分の根本的な原因を表しています3。最新の治療選択肢は患者の生存率を向上させましたが、根本的なメカニズムを標的とするのではなく、依然として主に対症療法です。したがって、これらの治療法の有効性は限られており、重篤な副作用を引き起こすことがよくあります4,5,6。現在の治療オプションを改善するには、根底にある病態生理学への洞察が必要であり、研究に適したモデルの必要性が生じます。小動物モデル、特にマウスモデルは、細胞の電気生理学、イオンチャネル機能、カルシウム処理に対する遺伝的影響など、不整脈形成の重要なメカニズムを研究できるため、不整脈研究において重要な役割を果たします7,8。
この目的のために、十分な量および生存率の単離された心房および心室心筋細胞が必要とされる。心房および心室筋細胞を得るための幅広い異なる単離アプローチが以前に説明されており9、10、11、12、13であり、一部のグループは、心房14または心室15のいずれかからのL型電流およびカルシウム電流誘発カルシウム過渡の同時測定からのデータを提示している。マウス心筋細胞。しかし、私たちの知る限り、1匹の動物からの心房および心室測定のデータはありません。研究者は、電気生理学からプロテオミクス、細胞収縮性やタンパク質相互作用などの機能研究、ミトコンドリア機能、遺伝学に至るまで、単離された心筋細胞を必要とする幅広いトピックに焦点を当てています。したがって、公開されているプロトコルの多くはパッチクランプ研究用に特別に開発されておらず、パッチクランプ研究には収量が制限され、細胞品質が不十分です。したがって、1匹の動物から単離された心房細胞と心室細胞のパッチクランプとカルシウム過渡の同時測定は、確立されたプロトコルでは実行できません。
パッチクランプ実験のためのマウス、特に心房筋細胞の単離は依然として困難です。本稿では、逆行性酵素ベースのランゲンドルフ灌流法により、高品質のマウス心房および心室筋細胞を単離するための簡単で迅速な方法を提供し、その後、1匹の動物からの正味の膜電流と電流誘発カルシウム過渡現象の両方を同時に測定することができます。この記事では、野生型マウスおよび遺伝子変異を有するマウスに由来する心房および心室筋細胞を単離するためのプロトコルについて詳述する。このプロトコルは、オスとメスのマウスに同様に使用できます。筋細胞単離、画像、および以下に説明する代表的な結果は、6(±1)ヶ月齢の野生型C57Bl/6マウスから得られた。それにもかかわらず、このプロトコルは、異なる遺伝子型を有する2〜24ヶ月の範囲の様々な年齢のマウスに首尾よく使用されている。 図1 は、分離セットアップと酵素灌流中のカニューレ心臓のクローズアップを示しています。

<table>
	<tbody>
		<tr>
			<td>2,3-Butanedione monoxime</td>
			<td>Sigma-Aldrich</td>
			<td>31550</td>
			<td></td>
		</tr>
		<tr>
			<td>27G cannula</td>
			<td>Servoprax</td>
			<td>L10220</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Aminopyridine</td>
			<td>Sigma-Aldrich</td>
			<td>A78403</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D12345</td>
			<td></td>
		</tr>
		<tr>
			<td>Aortic cannula</td>
			<td>Radnoti</td>
			<td>130163-20</td>
			<td></td>
		</tr>
		<tr>
			<td>BaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>342920</td>
			<td></td>
		</tr>
		<tr>
			<td>blunt surgical forceps</td>
			<td>Kent Scientific</td>
			<td>INS650915-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Calf Serum</td>
			<td>Sigma-Aldrich</td>
			<td>12133C</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C5080</td>
			<td></td>
		</tr>
		<tr>
			<td>Caffeine</td>
			<td>Sigma-Aldrich</td>
			<td>C0750</td>
			<td></td>
		</tr>
		<tr>
			<td>Circulating heated water bath</td>
			<td>Julabo</td>
			<td>ME</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type II</td>
			<td>Worthington</td>
			<td>LS994177</td>
			<td></td>
		</tr>
		<tr>
			<td>disscetion scissors</td>
			<td>Kent Scientific</td>
			<td>INS600124</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-aspartat K+-salt</td>
			<td>Sigma-Aldrich</td>
			<td>A2025</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo-3</td>
			<td>Invitrogen</td>
			<td>F3715</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo-3 AM</td>
			<td>Invitrogen</td>
			<td>F1242</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Guanosine 5&#8242;-triphosphate tris salt</td>
			<td>Sigma-Aldrich</td>
			<td>G9002</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating coil</td>
			<td>Radnoti</td>
			<td>158821</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Ratiopharm</td>
			<td>25.000 IE/5ml</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H9136</td>
			<td></td>
		</tr>
		<tr>
			<td>induction chamber</td>
			<td>CWE incorporated</td>
			<td>13-40020</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Cp-pharma</td>
			<td>1214</td>
			<td></td>
		</tr>
		<tr>
			<td>Jacketed heart chamber</td>
			<td>Radnoti</td>
			<td>130160</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Merck</td>
			<td>1049360250</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl x 6H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M0250</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4 x 7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M9397</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2ATP</td>
			<td>Sigma-Aldrich</td>
			<td>A2383</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4 x 2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>S5136</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon mesh (200 &#181;m)</td>
			<td>VWR-Germany</td>
			<td>510-9527</td>
			<td></td>
		</tr>
		<tr>
			<td>pasteur pipette</td>
			<td>Sigma Aldrich</td>
			<td>Z331759</td>
			<td></td>
		</tr>
		<tr>
			<td>petri-dishes</td>
			<td>Thermo Fisher</td>
			<td>150318</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic Acid F-127</td>
			<td>Sigma-Aldrich</td>
			<td>P2443</td>
			<td></td>
		</tr>
		<tr>
			<td>Probenecid</td>
			<td>Sigma-Aldrich</td>
			<td>P8761</td>
			<td></td>
		</tr>
		<tr>
			<td>Roller Pump</td>
			<td>Ismatec</td>
			<td>ISM597D</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical forceps</td>
			<td>Kent Scientific</td>
			<td>INS650908-4</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical scissors</td>
			<td>Kent Scientific</td>
			<td>INS700540</td>
			<td></td>
		</tr>
		<tr>
			<td>suturing silk</td>
			<td>Fine Science Tools</td>
			<td>NC9416241</td>
			<td></td>
		</tr>
		<tr>
			<td>syringe</td>
			<td>Merck</td>
			<td>Z683531-100EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurin</td>
			<td>Sigma-Aldrich</td>
			<td>86330</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of high quality murine atrial and ventricular myocytes for simultaneous measurements of ca2+ transients and l-type calcium current

マウスモデルは不整脈の研究において重要な役割を果たしており、イオンチャネル機能の変化やカルシウムの取り扱いなど、不整脈形成の重要なメカニズムを研究することができます。この目的のために、パッチクランプ測定を行ったり、カルシウム取り扱い異常を探ったりするために、高品質の心房または心室心筋細胞が必要です。しかしながら、現在の単離プロトコルによって得られた高品質の心筋細胞の限られた収量は、同じマウスで両方の測定を可能にすることができない。この記事では、逆行性酵素ベースのランゲンドルフ灌流を介して高品質のマウス心房および心室筋細胞を分離し、その後、1匹の動物からのカルシウム過渡現象とL型カルシウム電流を同時に測定する方法について説明します。マウスの心臓が得られ、大動脈を急速にカニューレに入れて血液を除去します。次に、心臓にカルシウムを含まない溶液(37°C)を灌流して、インターカレートされた椎間板のレベルで組織を解離させ、その後、カルシウムをほとんど含まない酵素溶液で細胞外マトリックスを破壊します(37°C)。消化された心臓はその後心房と心室に解剖されます。組織サンプルは細かく刻まれ、慎重に上下にピペッティングして溶解されます。酵素消化を停止し、細胞を生理学的カルシウム濃度まで段階的に再導入します。蛍光Ca2+指示薬をロードした後、単離された心筋細胞をカルシウム電流と過渡現象の同時測定用に調製します。さらに、分離の落とし穴が議論され、上記のように単離された心房および心室マウス筋細胞における同時カルシウム過渡測定を伴うパッチクランププロトコルおよびL型カルシウム電流の代表的な痕跡が提供されます。

単離収率は、カルシウム再導入後に10 μLの細胞懸濁液を顕微鏡スライド上にピペッティングすることによって決定されます。心房細胞単離の場合は100を超える生存可能な棒状の非収縮細胞/ 10 μL、心室細胞単離の場合は1,000を超える生存可能な棒状の非収縮細胞/ 10 μLが十分な収量と見なされ、一般的にこのプロトコルを使用して得られます。このプロトコルで得られた心房細胞は、心臓伝導?...

Watch this Scientific Journal Video about Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current at JoVE.com

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

マウスモデルは、不整脈形成の重要なメカニズムを研究することを可能にする。そのためには、パッチクランプ測定を行うために高品質の心筋細胞が必要です。ここでは、カルシウム一過性物質とL型カルシウム電流を同時に測定できる逆行性酵素ベースのランゲンドルフ灌流を介してマウス心房筋細胞と心室筋細胞を分離する方法について説明します。

Ca2+過渡現象とL型カルシウム電流の同時測定のための高品質マウス心房および心室筋細胞の分離

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

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Biology

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

2.9K ビュー.  Ludwig-Maximilians University Munich (LMU). マウスモデルは、不整脈形成の重要なメカニズムを研究することを可能にする。そのためには、パッチクランプ測定を行うために高品質の心筋細胞が必要です。ここでは、カルシウム一過性物質とL型カルシウム電流を同時に測定できる逆行性酵素ベースのランゲンドルフ灌流を介してマウス心房筋細胞と心室筋細胞を分離する方法について説明します。

ビデオ: Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

共焦点イメージングのための成人心筋細胞の単離と遺伝子操作

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

生物学

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

マウス心房と心室の心筋細胞における分離とKvチャンネル録音

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

The study of electrophysiological properties of cardiac ion channels with the patch-clamp technique and the exploration of cardiac cellular Ca2+ handling abnormalities requires isolated cardiomyocytes. In addition, the possibility to investigate myocytes from patients using these techniques is an invaluable requirement to elucidate the molecular basis of cardiac diseases such as atrial fibrillation (AF).1 Here we describe a method for isolation of human atrial myocytes which are suitable for both patch-clamp studies and simultaneous measurements of intracellular Ca2+ concentrations. First, right atrial appendages obtained from patients undergoing open heart surgery are chopped into small tissue chunks ("chunk method") and washed in Ca2+-free solution. Then the tissue chunks are digested in collagenase and protease containing solutions with 20 &mu;M Ca2+. Thereafter, the isolated myocytes are harvested by filtration and centrifugation of the tissue suspension. Finally, the Ca2+ concentration in the cell storage solution is adjusted stepwise to 0.2 mM. We briefly discuss the meaning of Ca2+ and Ca2+ buffering during the isolation process and also provide representative recordings of action potentials and membrane currents, both together with simultaneous Ca2+ transient measurements, performed in these isolated myocytes.

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

We describe the isolation of human atrial myocytes which can be used for intracellular Ca2+ measurements in combination with electrophysiological patch-clamp studies.

カルシウムの同時測定のための人間の心房筋細胞の分離<sup&gt; 2 +</sup&gt;過渡と膜電流

The study of  electrophysiological properties of cardiac ion channels with the patch-clamp  technique and the exploration of cardiac cellular Ca2+ ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

フローサイトメトリーによる腎上皮細胞における細胞質ゾルカルシウム測定

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

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 ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

ハイスループットマイクロプレート呼吸測定のためのマウスの骨格筋の最小量からミトコンドリアの単離

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

の開発と特性評価<em&gt;インビトロ</em&gt;微小血管ネットワークおよび内皮の定量的測定の[Ca<sup&gt; 2+</sup&gt;]<sub&gt;私</sub&gt;と一酸化窒素の生産

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

蛍光顕微鏡による生細胞内のミトコンドリアのカルシウムとミトコンドリア膜電位の同時測定

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

成体ラット心臓からの高品質心筋細胞、内皮細胞および線維芽細胞の同時単離

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of intracellular calcium release are the major intracellular calcium storage organelles, the endoplasmic reticulum (ER) and the more specialized sarcoplasmic reticulum (SR) in muscle cells. The dynamic release of calcium from these organelles is mediated by the ryanodine receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP3R) with refilling occurring through the sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. A genetically encoded calcium sensor (GECI) called CatchER was created to monitor the rapid calcium release from the ER/SR. Here, the detailed protocols for the transfection and expression of the improved, ER/SR-targeted GECI CatchER+ in HEK293 and C2C12 cells and its application in monitoring IP3R, RyR, and SERCA pump-mediated calcium transients in HEK293 cells using fluorescence microscopy is outlined. The receptor agonist or inhibitor of choice is dispersed in the chamber solution and the intensity changes are recorded in real time. With this method, a decrease in ER calcium is seen with RyR activation with 4-chloro-m-cresol (4-cmc), the indirect activation of IP3R with adenosine triphosphate (ATP), and inhibition of the SERCA pump with cyclopiazonic acid (CPA). We also discuss protocols for determining the in situ Kd and quantifying basal [Ca2+] in C2C12 cells. In summary, these protocols, used in conjunction with CatchER+, can elicit receptor mediated calcium release from the ER with future application in studying ER/SR calcium related pathologies.

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

We present protocols for the application of our targeted genetically-encoded calcium indicator (GECI) CatchER+ for monitoring rapid calcium transients in the endoplasmic/sarcoplasmic reticulum (ER/SR) of HEK293 and skeletal muscle C2C12 cells using real-time fluorescence microscopy. A protocol for the in situ Kd measurement and calibration is also discussed.

目標とするCaを用いたER / SRカルシウム放出のモニタリング<sup&gt; 2+</sup&gt;センサーキャッチャー<sup&gt; +</sup

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of ...