著者らは、ポルトガル科学技術財団(FCT)、欧州連合、クエロ・デリメンシア・エストラテジコ・ナシオナル(QREN)、フンド・ヨーロッパ・デ・デセンボルヴィメント地域(FEDER)、プログラム・オペラシオナル・ファクタレス・デ・コンペティティビダード(COMPETE)に感謝する。このプロジェクトは、COMPETEERによってサポートされています COMPETE2020 - プログラムオペラシオナルコンペティティビダーデEインテルナシオナリザサン(POCI)、プロジェクトDOCNET(NORTE-01-0145-FEDER-000003)、ノルテポルトガル地域運用プログラム(NORTE 2020)、ポルトガル2020パートナーシップの下で、合意、 欧州地域開発基金(ERDF)を通じて、リスボンの地域運用プログラムである欧州構造投資ファンドが支援するプロジェクトNETDIAMOND(POCI-01-0145-FEDER-016385)を通じて行います。パティシア・ロドリゲスはFCT(SFRH/BD/96026/2013)によって資金提供され、ジョアン・アルメイダ=コエーリョはユニバーシダード・ド・ポルト/FMUPとFSEフンド・ソーシャル・ヨーロッパによって資金提供されました。 NORTE 2020-プログラムオペラシオナル地域ドノルテ、(NORTE-08-5369-FSE-000024-プログラム・ドゥートレ)。

<ol>
	<li>Leite-Moreira, A. 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=Stretch-induced+compliance:+a+novel+adaptive+biological+mechanism+following+acute+cardiac+load.">Stretch-induced compliance: a novel adaptive biological mechanism following acute cardiac load.</a> Cardiovascular Research. 114 (5), 656-667 (2018).</li><li>Falcao-Pires, I., Fontes-Sousa, A. P., Lopes-Conceicao, L., Bras-Silva, C., Leite-Moreira, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+myocardial+stiffness+by+&#946;-adrenergic+stimulation+-+its+role+in+normal+and+failing+heart.">Modulation of myocardial stiffness by &#946;-adrenergic stimulation - its role in normal and failing heart.</a> Physiological Research. 60 (4), 599-609 (2011).</li><li>Cokkinos, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Introduction to Translational Cardiovascular Research. , 371-387 (2015).</li><li>van der Velden, J., Stienen, G. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+Disorders+and+Pathophysiology+of+Sarcomeric+Proteins.">Cardiac Disorders and Pathophysiology of Sarcomeric Proteins.</a> Physiological Reviews. 99 (1), 381-426 (2019).</li><li>Garnier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attachment+procedures+for+mechanical+manipulation+of+isolated+cardiac+myocytes:+a+challenge.">Attachment procedures for mechanical manipulation of isolated cardiac myocytes: a challenge.</a> Cardiovascular Research. 28 (12), 1758-1764 (1994).</li><li>Brady, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+isolated+cardiac+myocytes.">Mechanical properties of isolated cardiac myocytes.</a> Physiological Reviews. 71 (2), 413-428 (1991).</li><li>Falcao-Pires, I., Leite-Moreira, A. F., Cokkinos, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+20.">Chapter 20.</a> Introduction to Translational Cardiovascular Research. 20, 371-387 (2015).</li><li>Liang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Teaching+calcium-induced+calcium+release+in+cardiomyocytes+using+a+classic+paper+by+Fabiato.">Teaching calcium-induced calcium release in cardiomyocytes using a classic paper by Fabiato.</a> Advances Physiology Education. 32 (1), 1-10 (2008).</li><li>Roche, S. M., Gumucio, J. P., Brooks, S. V., Mendias, C. L., Claflin, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Maximum+Isometric+Force+Generated+by+Permeabilized+Skeletal+Muscle+Fibers.">Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers.</a> Journal of Visualized Experiments. (100), e52695 (2015).</li><li>Huxley, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+structure+and+theories+of+contraction.">Muscle structure and theories of contraction.</a> Progress Biophysics and Biophysical Chemistry. 7, 255-318 (1957).</li><li>Sequeira, 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=Synergistic+role+of+ADP+and+Ca(2+)+in+diastolic+myocardial+stiffness.">Synergistic role of ADP and Ca(2+) in diastolic myocardial stiffness.</a> Journal Physiology. 593 (17), 3899-3916 (2015).</li><li>Edes, I. F., 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=Rate+of+tension+redevelopment+is+not+modulated+by+sarcomere+length+in+permeabilized+human,+murine,+and+porcine+cardiomyocytes.">Rate of tension redevelopment is not modulated by sarcomere length in permeabilized human, murine, and porcine cardiomyocytes.</a> American Journal Physiology Regulatory Integrative Comparative Physiology. 293 (1), R20-R29 (2007).</li><li>King, N. 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=Mouse+intact+cardiac+myocyte+mechanics:+cross-bridge+and+titin-based+stress+in+unactivated+cells.">Mouse intact cardiac myocyte mechanics: cross-bridge and titin-based stress in unactivated cells.</a> Journal General Physiology. 137 (1), 81-91 (2011).</li><li>Hamdani, 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=Myocardial+titin+hypophosphorylation+importantly+contributes+to+heart+failure+with+preserved+ejection+fraction+in+a+rat+metabolic+risk+model.">Myocardial titin hypophosphorylation importantly contributes to heart failure with preserved ejection fraction in a rat metabolic risk model.</a> Circulation Heart Failure. 6 (6), 1239-1249 (2013).</li><li>Miranda-Silva, D., 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=Characterization+of+biventricular+alterations+in+myocardial+(reverse)+remodelling+in+aortic+banding-induced+chronic+pressure+overload.">Characterization of biventricular alterations in myocardial (reverse) remodelling in aortic banding-induced chronic pressure overload.</a> Scientific Reports. 9 (1), 2956 (2019).</li><li>Rodrigues, P. G., 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=Early+myocardial+changes+induced+by+doxorubicin+in+the+nonfailing+dilated+ventricle.">Early myocardial changes induced by doxorubicin in the nonfailing dilated ventricle.</a> American Journal Physiology Heart Circulatory Physiology. 316 (3), H459-H475 (2019).</li><li>van der Velden, 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=Alterations+in+myofilament+function+contribute+to+left+ventricular+dysfunction+in+pigs+early+after+myocardial+infarction.">Alterations in myofilament function contribute to left ventricular dysfunction in pigs early after myocardial infarction.</a> Circulation Research. 95 (11), e85-e95 (2004).</li><li>Wakili, 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=Multiple+potential+molecular+contributors+to+atrial+hypocontractility+caused+by+atrial+tachycardia+remodeling+in+dogs.">Multiple potential molecular contributors to atrial hypocontractility caused by atrial tachycardia remodeling in dogs.</a> Circulation: Arrhythmia Electrophysiology. 3 (5), 530-541 (2010).</li><li>Ait Mou, Y., le Guennec, J. Y., Mosca, E., de Tombe, P. P., Cazorla, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+contribution+of+cardiac+sarcomeric+proteins+in+the+myofibrillar+force+response+to+stretch.">Differential contribution of cardiac sarcomeric proteins in the myofibrillar force response to stretch.</a> Pflugers Archiv. 457 (1), 25-36 (2008).</li><li>Falcao-Pires, I., 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=Diabetes+mellitus+worsens+diastolic+left+ventricular+dysfunction+in+aortic+stenosis+through+altered+myocardial+structure+and+cardiomyocyte+stiffness.">Diabetes mellitus worsens diastolic left ventricular dysfunction in aortic stenosis through altered myocardial structure and cardiomyocyte stiffness.</a> Circulation. 124 (10), 1151-1159 (2011).</li><li>Lim, C. C., 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=Anthracyclines+induce+calpain-dependent+titin+proteolysis+and+necrosis+in+cardiomyocytes.">Anthracyclines induce calpain-dependent titin proteolysis and necrosis in cardiomyocytes.</a> Journal Biology Chemistry. 279 (9), 8290-8299 (2004).</li><li>Woulfe, K. C., 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=A+Novel+Method+of+Isolating+Myofibrils+From+Primary+Cardiomyocyte+Culture+Suitable+for+Myofibril+Mechanical+Study.">A Novel Method of Isolating Myofibrils From Primary Cardiomyocyte Culture Suitable for Myofibril Mechanical Study.</a> Frontiers Cardiovascular Medicine. 6, 12 (2019).</li><li>Ait Mou, Y., Bollensdorff, C., Cazorla, O., Magdi, Y., de Tombe, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+cardiac+biophysical+properties.">Exploring cardiac biophysical properties.</a> Global Cardiology Science Practice. 2015, 10 (2015).</li></ol>

著者には利益相反はない。

皮膚付き心筋細胞を用いた心機能のインビトロ評価は、生理学的(例えば、ストレッチ)および病的文脈(例えば虚血)において心筋細胞レベルで起こる修飾を明らかにするための重要な技術を表す。この方法論は、解凍されたサンプルから得られた心筋細胞の機能を評価するために最小限の量の心筋を必要とするなどのいくつかの利点を有する。広範囲の種(マウス13、ラット1、14、15、ウサギ16、ブタ17、犬18、モルモット19およびヒト20)および心房、左および右心室または梗塞心臓の特定の領域を含む異なる心臓の位置を使用して、心筋細胞を使用する。さらに、この技術は、Ca2+およびエネルギー(ATP)の特定の濃度を提供しながら、そのネイティブ構成における規制および収縮構造の機能を測定することを可能にする。
この手法のシンプルさにもかかわらず、いくつかの重要な手順があります。サンプルコレクションを含め、最初から各ステップの品質を保証することが不可欠です。ミオフィラメントタンパク質はプロテアーゼ21に感受性である。したがって、収集直後に液体窒素にサンプルを保存することが義務付けられています。以前は凍結されていなかった新鮮なサンプルは、有意に高い力を発生させるので、同じプロトコルで新鮮なサンプルと凍結したサンプルで行われた測定を混合することはお勧めできません。2番目に重要なステップは、心筋細胞の抽出です。この手順の間、ほとんどの場合、氷上のサンプルを維持することが重要です。プロテアーゼ阻害剤カクテルは、抽出/透過性22中のタンパク質分解のリスクを低減するために使用することができる。第三に、このステップが無視されたときに質の高い心筋細胞を減らすことに注意したので、サンプルは正確なメスの動きを使用して小さな部分にカットする必要があります。もう一つの重要なステップは、トリトンを洗浄すること(細胞を透過させるが、その不活性化を促進する)とできるだけ多くの細胞を上清に保つこととの間の適切なバランスを持つことが困難であるため、心筋細胞を洗浄することです。まず、抽出と各サンプル、種またはプロトコルの洗い流しの数を試してみることが重要です。例えば、私たちの手の中で、ZSF1肥満ラット組織抽出は「脂肪」の側面を持っており、接着中にこれらの細胞はより滑りやすいが、測定することはより困難ではないことを指摘した。この問題を回避する方法は、動物1匹につき妥当な数の細胞を持つために、より多くの実験を行うことでした。さらに、良好な線条と妥当な長さで、接着する良いセルを選択することが重要です。心筋細胞がこれらの特徴を持たない場合、それは主に針の先端から切り離されるか、または無/低力を開発する。また、接着の時間と細胞を針に接着する有効性を考慮して、心筋細胞の付着に正しい接着剤を使用することも重要です。私たちの手の中で、シリコーン接着剤(材料のテーブル)は速く(10-15分)、十分に強く治ります。最後の重要なステップは、細胞を接着した後5分の心筋細胞を慎重に持ち上げ(細胞をカバースリップに接着することを避けるため)、ウェルに移動する前に(顕微鏡ステージで引きずられる細胞を避けるために)関連しています。 表 7 に、この手法に関連するトラブルシューティング、その根本的な原因、および頻繁な問題を解決するための可能な解決策をまとめます。
この方法の主な制限は、myofilamentの収縮性に関連するすべての質問(myofilamentが活性化/非アクティブ化する速度など)に答えることができないということです。in vivo設定では、細胞内脱分極、細胞内Ca2+ の増加および筋糸状細胞への拡散が収縮するために起こる必要があるのに対し、皮膚付き心筋細胞ではCa2+ の筋糸に対する拡散は、細胞がCa2+ 溶液に沈下されるとすぐに起こる。このより速い速度のCa2+ 拡散は、ミオブフィラメントの活性化/非活性化分析23にバイアスを与える。
これらの実験は、温度、溶液pH、機械的摂動(緩み再伸縮対緩み)および細胞付着手順(ピンタイ対接着剤)を含む異なる要因の影響を受けるが、これらの変数の全てはktrの点で文献の不一致を説明し、サルコメアの長さ依存的な力4,12の増加を示す。
この技術の将来の進歩には、透過性心筋細胞ではなく無傷で機能研究を行うすることが含まれる。この技術は、新たに単離された心筋細胞(以前は凍結されていない)に頼るという欠点を有する。この方法論に直接関係しない別の重要な問題は、それが大きく影響する可能性があり、それはサンプル凍結ストレージの最大期間に関連しています。具体的には、保存時間を通じてmyofilament分解の程度を確立することが必須である(すなわち、抽出された心筋細胞から導出された良好な品質の機能データを保証するために、凍結したサンプルを保存できる期間)。

従来、心筋の機械的特性の評価は、主に乳頭筋や柱状筋筋1,2などの多細胞製剤において試みられてきた。多細胞性心筋ストリップには、異種細胞集団が含まれ、その中には、オリエンテーションと力の発生、電気活動およびストレス/歪み分布、ならびに周囲の結合組織マトリックス3,4を含む収縮性心筋細胞が含まれる。コラーゲンを含まず、単一の心筋細胞を含む調製物は、サルコメア長さおよび交差ブリッジ収縮特性を非常に正確かつ制御された方法で測定することを可能にする5,6.したがって、過去40年間に、いくつかの方法論が開発され、単一の心筋細胞6,7の機械的、収縮性、および緩和特性を調査できる。これらの細胞の収縮機能は、サルコメア長さおよびクロスブリッジサイクリングキネティクス3に強く依存している。したがって、サルコメアの長さや性能、ならびにクロスブリッジ機能および収縮特性を評価することを考慮して、単一の単一の単一の心臓細胞で直接筋肉機能を調査することが望ましい。しかし、μNレベルでの力測定を記録しながら、合理的な光学サルコメア分解能を有する機能的心筋細胞の単離および取り付けは、依然として困難であり、3,6.その他の課題は、新たに採取した生検から心筋細胞を分離するために設置する必要がある物流です。例えば、ヒト生検コレクションの予測不可能性は、実験の実現可能性を危険にさらす可能性があります。
さらに、科学的手順(3Rの原理)のための動物実験の置き換え、削減および改良に関する倫理的な懸念は、細胞および組織レベル、好ましくはヒト生検、またはより小さな動物サンプルでの研究の変化を促進している。実際に、より小さなレベルの複雑さの中でインビトロで心機能を評価するための方法論の進歩的な改良は、結果を全身に適切に統合し、臨床シナリオ7に翻訳することを可能にする。全体として、-80°Cで保存されたサンプルを使用して心筋細胞を抽出することは魅力的な代替手段であり得る。
心筋組織は小片に切断され、モルタルと害虫で均質化される。この均質化の結果は、皮質変化の損傷を伴う皮の束と単離された細胞の懸濁液であり、そこで近視が入浴媒体にさらされ、すべての細胞成分が洗い流される。サルコレンマから遠く離れた筋線維のような構造は保存される。従って、筋細動装置に関連するサルコメアの短縮および機能的特性はそのまま保持され、8,9を記録することができる。
心筋細胞力測定システムは、心筋細胞長の調整に用いられる電磁モータと、等角心筋細胞収縮を測定する力トランスデューサーから構成されています。透過性、または皮をむいた心筋細胞は、リラックスした溶液([Ca 2+]<10nM)を含む実験室に配置され、シリコン接着は2本の細い針に接着され、1つはモーターに取り付けられ、もう1つは力トランスデューサに取り付けられる。心筋細胞の形態とサルコメアの長さを決定するために光学系が使用されます。実験プロトコルは、多くの場合、異なるCa2+濃度を含むバッファ溶液上の一連の力記録、アクチンミオシン架橋運動学の決定、およびあらかじめ定義されたサルコメア長さでの取り付けられた心筋細胞の受動的緊張の測定からなる(図1)。液体窒素で凍結した心筋試料からの透過性心筋細胞の分離(そしてその後-80°Cで保存される)は、細胞力学とタンパク質生化学を利用して断面積当たりの最大Ca2+活性化(活性)力(T∙活性、kN m-2)を測定する技術です。 受動)緊張(Tパッシブ、kN∙m-2)、筋ヒフィラメントCa2+感度(pCa50)、筋ヒフィラメント協同率(nヒル)、力再発達率(ktr)、ならびにTアクティブのサルコメア長依存性、Tパッシブ、pCa50、nHillおよびktr。
このプロトコルの目的は、異なる種の凍結サンプルから分離された単一の皮膚心筋細胞の機能的機械的特性を評価する信頼できる手順として、心筋細胞力測定システムの可能性を説明し、要約することです。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetone</td>
			<td>Sigma</td>
			<td>34580</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#8217;-triphosphate disodium salt hydrate (Na2ATP)</td>
			<td>Sigma</td>
			<td>A2383</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium carbonate (CaCO3)</td>
			<td>Merck</td>
			<td>1.02067.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>VWR</td>
			<td>24720.157</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate (MgCl2.6H2O)</td>
			<td>Merck</td>
			<td>1.05833.0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride solution (MgCl2 1M)</td>
			<td>Sigma</td>
			<td>M1028</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Bis(2-hydroxyethyl)taurine (BES)</td>
			<td>Sigma</td>
			<td>B9879</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphocreatine dissodium salt hydrate (Na2PCr)</td>
			<td>Sigma</td>
			<td>P7936</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Merck</td>
			<td>1.04936.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium hydroxide (KOH)</td>
			<td>Merck</td>
			<td>8.14353.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Propionic acid (C3H6O2)</td>
			<td>Merck</td>
			<td>8.00605.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone Squeeze Tube</td>
			<td>Marineland</td>
			<td>31003</td>
			<td></td>
		</tr>
		<tr>
			<td>Tritiplex (EGTA)</td>
			<td>Merck</td>
			<td>1.08435.0025</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton&#174; X-100 10%</td>
			<td>Merck</td>
			<td>648463</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue homogeneizer (GKH GT Motor Control)</td>
			<td>Terre Haute Glas-col</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Length Controller ( Model 315C-I)</td>
			<td>Aurora Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Force Transducer (Model 403 A)</td>
			<td>Aurora Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software ASI 600A</td>
			<td>Aurora Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sotware VSL (Model 900B)</td>
			<td>Aurora Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope (IX51)</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro assessment of cardiac function using skinned cardiomyocytes

本稿では、単一の透過性(「皮をむいた」)心筋細胞を分離し、力測定装置とモーターに取り付けて機能研究を行うために必要な手順について説明します。これらの研究は、心筋細胞の剛性(受動的な力)とその異なるカルシウム(Ca2+)含有溶液による活性化を測定することを可能にする:最大力の発達、筋状放射状Ca2+感受性(pCa50)、協同性(nHill)および力再発達率(ktr)を決定する。この方法はまた、筋膜に直接作用する薬物の効果と、心筋細胞の活性および受動的特性の両方に対する外因性組換えタンパク質の発現の決定を可能にする。臨床的に、皮膚の心筋細胞研究は、多くの心筋疾患の病態生理を強調し、筋膜を標的とする治療介入の影響のインビトロ評価を可能にする。全体として、この技術は、開胸手術または移植手術中に得られた動物モデルとヒト組織におけるin vitroおよびin vivoパラメータの間の相関関係を調査することによって、心臓病理生理学の解明を可能にする。

Watch this Scientific Journal Video about In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes at JoVE.com

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

このプロトコルは、皮をむいた心筋細胞を用いた心機能の抽出および評価の技術を段階的に記述することを目的とする。この方法論は、マウスから男性まで、異なる心臓の場所から収集することができる小さな凍結生検を使用して、myofilament機能の測定および急性調節を可能にする。

皮膚心筋細胞を用いた心機能のインビトロ評価

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring ...

in-vitro-assessment-of-cardiac-function-using-skinned-cardiomyocytes

Research

JoVE Journal

Medicine

5.9K ビュー.  Universidade do Porto. このプロトコルは、皮をむいた心筋細胞を用いた心機能の抽出および評価の技術を段階的に記述することを目的とする。この方法論は、マウスから男性まで、異なる心臓の場所から収集することができる小さな凍結生検を使用して、myofilament機能の測定および急性調節を可能にする。

ビデオ: In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of cardiovascular disease. Although molecular biology is necessary in identifying key changes in the signaling pathway, it is not a surrogate for functional significance. While physiology can provide answers to the question of function, combining physiology with biochemical assessment of metabolites in the intact, beating heart allows for a complete picture of cardiac function and energetics. For years, our laboratory has utilized isolated heart perfusions combined with nuclear magnetic resonance (NMR) spectroscopy to accomplish this task. Left ventricular function is assessed by Langendorff-mode isolated heart perfusions while cardiac energetics is measured by performing 31P magnetic resonance spectroscopy of the perfused hearts. With these techniques, indices of cardiac function in combination with levels of phosphocreatine and ATP can be measured simultaneously in beating hearts. Furthermore, these parameters can be monitored while physiologic or pathologic stressors are instituted. For example, ischemia/reperfusion or high workload challenge protocols can be adopted. The use of aortic banding or other models of cardiac pathology are apt as well. Regardless of the variants within the protocol, the functional and energetic significance of molecular modifications of transgenic mouse models can be adequately described, leading to new insights into the associated enzymatic and metabolic pathways. Therefore, 31P NMR spectroscopy in the isolated perfused heart is a valuable research technique in animal models of cardiovascular disease.

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Langendorff-mode isolated heart perfusion, in conjunction with 31P NMR spectroscopy, combines the fields of biochemistry and physiology into one experiment. The protocol allows for the dynamic measurement of high energy phosphate content and turnover in the heart while concurrently monitoring physiologic function. When performed correctly, this is a valuable technique in the assessment of cardiac energetics.

使用する絶縁マウスハーツの心機能およびエネルギー論の評価 31 P NMR分光法

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of ...

医学

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery (SALLI) MRI in Rats

Small animal magnetic resonance imaging is an important tool to study cardiac function and changes in myocardial tissue. The high heart rates of small animals (200 to 600 beats/min) have previously limited the role of CMR imaging. Small animal Look-Locker inversion recovery (SALLI) is a T1 mapping sequence for small animals to overcome this problem 1. T1 maps provide quantitative information about tissue alterations and contrast agent kinetics. It is also possible to detect diffuse myocardial processes such as interstitial fibrosis or edema 1-6. Furthermore, from a single set of image data, it is possible to examine heart function and myocardial scarring by generating cine and inversion recovery-prepared late gadolinium enhancement-type MR images 1.
	The presented video shows step-by-step the procedures to perform small animal CMR imaging. Here it is presented with a healthy Sprague-Dawley rat, however naturally it can be extended to different cardiac small animal models.

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery SALLI MRI in Rats

Contrast enhanced cardiac magnetic resonance (CMR) imaging allows comprehensive in vivo assessment of the heart in small animal models of cardiovascular disease. Here we detail the procedures to perform CMR imaging, reconstruction and analysis using Small Animal Lock-Locker Inversion Recovery (SALLI) in rats.

小動物ルックロッカー反転回復（サリー）ラットにおけるMRIを用いた心機能と心筋形態の評価

Small animal magnetic resonance  imaging is an important tool to study cardiac function and changes in myocardial  tissue. The high heart rates of small ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

経胸壁心エコー検査による肺動脈狭窄のマウスモデルにおける右室の構造と機能の評価

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

Transcutaneous Assessment of Renal Function in Conscious Rodents

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and time-consuming. In addition, serial blood or urine samples are required, with the associated stress for the experimental animals. A recent technique significantly reduces the investment in time and resources, minimizing the invasiveness and the animal stress, but being equally valid as the traditional approaches. The method measures transcutaneously renal function. Using an optical device and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin, this technique is capable of measuring the elimination kinetics of the marker through the skin. With neither blood nor urine samples nor the associated laboratory assays needed, the results of the transcutaneous measurement are almost instantaneously available. The method has been already validated in different species and successfully applied in several models of renal pathology. Moreover, due to its minimally invasive characteristics, it is suitable for sequential measurements within the same animal. Here is provided a detailed protocol to carry out the transcutaneous assessment of renal function in rodents.

Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

コンシャスげっ歯類における腎機能の経皮評価

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

準備と<em&gt;インビトロ</em磁化のmiR修飾内皮細胞の&gt;キャラクタリゼーション

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

顕微鏡を用いたひと脂肪組織血管機能評価

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation time and increases the risk of adverse post-transplantation outcomes. Moreover, the static nature of CS does not allow for organ evaluation or intervention during the preservation interval. Normothermic ex situ heart perfusion (ESHP) is a novel method for preservation of the donated heart that minimizes cold ischemia by providing oxygenated, nutrient-rich perfusate to the heart. ESHP has been shown to be non-inferior to CS in the preservation of standard-criteria donor hearts and has also facilitated the clinical transplantation of the hearts donated after the circulatory determination of death. Currently, the only available clinical ESHP device perfuses the heart in an unloaded, non-working state, limiting assessments of myocardial performance. Conversely, ESHP in working mode provides the opportunity for comprehensive evaluation of cardiac performance by assessment of functional and metabolic parameters under physiologic conditions. Moreover, earlier experimental studies have suggested that ESHP in working mode may result in improved functional preservation. Here, we describe the protocol for ex situ perfusion of the heart in a large mammal (porcine) model, which is reproducible for different animal models and heart sizes. The software program in this ESHP apparatus allows for real-time and automated control of the pump speed to maintain desired aortic and left atrial pressure and evaluates a variety of functional and electrophysiological parameters with minimal need for supervision/manipulation.

Normothermic ex situ heart perfusion (ESHP), preserves the heart in a beating, semi-physiologic state. When performed in a working mode, ESHP provides the opportunity to perform sophisticated assessments of donor heart function and organ viability. Here, we describe our method for myocardial performance evaluation during ESHP.

常温動作モードの場心の灌流 Ex: 心機能と代謝の評価

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation ...

Echocardiographic Assessment of Cardiac Anatomy and Function in Adult Rats

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional imaging to study the cardiac anatomy of the left ventricle and M-mode echo to assess its dimensions. However, this could limit a comprehensive study. Herein, we describe a protocol that allows an assessment of the heart chamber size, left ventricular function (systolic and diastolic) and valvular function. A conventional medical ultrasound machine was used in this protocol and different echo views were obtained through left parasternal, apical and suprasternal windows. In the left parasternal window, the long and short axis were acquired to analyze left chamber dimensions, right ventricle and pulmonary artery dimensions, and mitral, pulmonary and aortic valve function. The apical window allows the measurement of heart chamber dimensions and evaluation of systolic and diastolic parameters. It also allows Doppler assessment with detection and quantification of heart valve disturbances (regurgitation or stenosis). Different segments and walls of the left ventricle are visualized throughout all views. Finally, the ascending aorta, aortic arch, and descending aorta can be imaged through the suprasternal window. A combination of ultrasound imaging, Doppler flow and tissue Doppler assessment have been obtained to study cardiac morphology and function. This represents an important contribution to improve the assessment of cardiac function in adult rats with impact for research using these animal models.

A non-invasive protocol for transthoracic echocardiography assessment of cardiac anatomy and function for adult rats is presented in the current study. The heart valves, all four cardiac chambers and the ascending aorta, aortic arch and descending aorta are studied in detail.

成人ラットにおける心臓解剖学と機能の心エコー評価

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

移植質評価のための化学バイオプシーの使用

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...

Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily and highly reproducible MI induction procedure is required to obtain further insight and better understanding of the underlying pathological changes. This procedure can also be used to evaluate the effects or potency of new and promising treatments (as drugs or interventions) in acute MI, subsequent remodeling and heart failure (HF). After intubation and pre-operative preparation of the animal, an anesthetic protocol with isoflurane was performed, and the surgical procedure was conducted&#160;quickly. Using a minimally invasive approach, the left anterior descending artery (LAD) was located and occluded by a ligature. The occlusion can be performed acutely for subsequent reperfusion (ischemia/reperfusion injury). Alternatively, the vessel can be ligated permanently to investigate the development of chronic MI, remodeling or HF. Despite common pitfalls, the drop-out rates are minimal. Various treatments such as remote ischemic conditioning can be examined for their cardioprotective potential pre-, peri- and post-operatively. The post-operative recovery was quick as the anesthesia was precisely controlled and the duration of the operation was short. Post-operative analgesia was administered for three days. The minimally invasive procedure reduces the risk of infection and inflammation. Furthermore, it facilitates rapid recovery. The &#8220;working heart&#8221; measurements were performed ex vivo and enabled precise control of preload, afterload and flow. This procedure requires specific equipment and training for adequate performance. This manuscript provides a detailed step-by-step introduction for conducting these measurements.

This article presents an efficient method to perform myocardial ischemia and subsequent chronic reperfusion in rats using a minimally invasive approach. In addition, left ventricular hemodynamic function of rats is assessed by echocardiography and isolated working heart methods.&#160;

ラットの心筋梗塞を誘導する半最小侵襲法と、単離した働く心臓システムによる心機能の評価

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...