Les auteurs remercient la Fondation portugaise pour la science et la technologie (FCT), l’Union européenne, Quadro de Referência Estratégico Nacional (QREN), Fundo Europeu de Desenvolvimento Regional (FEDER) et Programa Operacional Factores de Competitividade (COMPETE) pour le financement de l’unité de recherche unic (UID/IC/00051/2013). Ce projet est soutenu par FEDER dans le cadre de COMPETE 2020 - Programa Operacional Competitividade E Internacionalização (POCI), le projet DOCNET (NORTE-01-0145-FEDER-000003), soutenu par le programme opérationnel régional Norte Portugal (NORTE 2020), dans le cadre de l’accord de partenariat Portugal 2020, par l’intermédiaire du Fonds européen de développement régional (FEDF), le projet NETDIAMOND (POCI-01-0145-FEDER-016385), soutenu par les Fonds structurels et d’investissement européens, programme opérationnel régional de Lisbonne 2020. Patrícia Rodrigues a été financée par fct (SFRH/BD/96026/2013) et João Almeida-Coelho a été par Universidade do Porto/FMUP et FSE-Fundo Social Europeu, NORTE 2020-Programa Operacional Regional do Norte, (NORTE-08-5369-FSE-000024-Programas Doutorais).

<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>

Les auteurs n’ont aucun conflit d’intérêts.

L’évaluation in vitro de la fonction cardiaque à l’aide de cardiomyocytes écorchés représente une technique importante pour clarifier les modifications qui se produisent au niveau des cardiomyocytes dans le contexte physiologique (p. ex., étirement) et pathologique (p. ex., ischémie). Cette méthodologie présente plusieurs avantages tels que l’exigence d’une quantité minimale de myocarde pour évaluer la fonction dans les cardiomyocytes obtenus à partir d’échantillons décongelés; en utilisant des cardiomyocytes d’un large éventaild’espèces (souris 13, rat1,14,15, lapin16, porc17, chien18, cobaye19 et humain20) et différents endroits cardiaques, y compris les atria, ventricules gauche et droit ou une région spécifique du cœur infarctus. De plus, cette technique permet d’offrir des concentrations spécifiques de Ca2+ et d’énergie (ATP) tout en mesurant la fonction des structures réglementaires et contractiles dans leur configuration native.
Malgré la simplicité de cette technique, il y a quelques étapes critiques. Il est essentiel de garantir la qualité de chaque étape dès le début, y compris la collecte d’échantillons. Les protéines de myofilament sont sensibles aux protéases21. Il est donc obligatoire de stocker les échantillons dans de l’azote liquide immédiatement après sa collecte. Les échantillons frais, qui n’étaient pas congelés auparavant, développeront des forces significativement plus élevées, de sorte qu’il n’est pas conseillé de mélanger la mesure effectuée dans des échantillons frais et congelés dans le même protocole. La deuxième étape la plus critique est l’extraction des cardiomyocytes. Au cours de cette procédure, il est crucial de maintenir l’échantillon sur la glace la plupart du temps. Un cocktail inhibiteur de la protéase peut être utilisé pour réduire le risque de dégradation des protéines pendant l’extraction/perméabilisation22. Troisièmement, les échantillons devraient être coupés en petits morceaux à l’aide de mouvements précis du scalpel puisque nous avons noté des cardiomyocytes de qualité réduite lorsque cette étape a été ignorée. Une autre étape critique est de laver les cardiomyocytes car il est difficile d’avoir le bon équilibre entre laver Triton (perméabilize la cellule, mais favorise son déglutage) et de garder autant de cellules dans le supernatant que possible. Il est important d’essayer d’abord l’extraction et le nombre de lavages pour chaque échantillon, espèce ou protocole. Par exemple, dans nos mains, nous avons noté que les extractions obèses de tissu de rat de ZSF1 ont un aspect « gras », qui a rendu ces cellules plus glissantes pendant le collage mais pas plus difficiles à mesurer. La façon dont nous contournons ce problème était d’effectuer plus d’expériences pour avoir un nombre raisonnable de cellules par animal. En outre, il est crucial de choisir une bonne cellule à coller, à savoir avec une bonne striation et une longueur raisonnable. Si le cardiomyocyte n’a pas ces dispositifs, il se détachera la plupart du temps des bouts d’aiguille ou développera aucune/basse force. Il est également important d’utiliser la colle correcte pour l’attachement cardiomyocyte, compte tenu du temps de collage et de son efficacité pour coller la cellule à l’aiguille. Dans nos mains, la colle de silicone (Table of Materials) guérit rapidement (10-15 min) et assez fort. Enfin, la dernière étape critique est liée au levage soigneusement du cardiomyocyte 5 min après collage de la cellule (pour éviter de coller la cellule à la couverture) et avant de la déplacer vers les puits (pour éviter que la cellule ne soit traînée par l’étape du microscope). Le tableau 7 résume le dépannage associé à cette technique, ses causes sous-jacentes et les solutions possibles pour surmonter les problèmes fréquents.
La principale limitation de cette méthode est qu’elle ne peut pas répondre à toutes les questions liées à la contractilité du myofilament, telles que la vitesse à laquelle les myofilaments s’activent/se désactivent. Dans le cadre in vivo, la dépolarisation membranaire, l’augmentation intracellulaire ca2+ et sa diffusion aux myofilaments doivent se produire pour que les myocytes se contractent, tandis que dans les cardiomyocytes écorchés Ca2+ diffusion aux myofilaments se produit immédiatement lorsque la cellule est submergée dans la solution Ca2+. Ce taux plus rapide de diffusion Ca2+ biaisera l’analyse d’activation/désactivation des myofilaments23.
Ces expériences sont influencées par différents facteurs, y compris la température, le pH de solution, la perturbation mécanique (slack-re-stretch vs slack) et les procédures de fixation cellulaire (attache d’épingle vs colle), toutes ces variables tenant compte des écarts de littérature en termes de ktr et de l’augmentation dépendante de la longueur du sarcomere en force4,12.
Les progrès futurs de la technique incluent l’exécution d’études fonctionnelles dans des cardiomyocytes intacts plutôt que perméabilisés. Cette technique a l’inconvénient de s’appuyer sur des cardiomyocytes fraîchement isolés (non préalablement congelés). Une autre question importante qui n’est pas directement liée à cette méthodologie, mais qui pourrait avoir une incidence importante sur celle-ci, est liée à la période maximale d’entreposage congelé de l’échantillon. Plus précisément, il est obligatoire d’établir le degré de dégradation du myofilament tout au long du temps d’entreposage (c.-à-d. pendant combien de temps les échantillons congelés peuvent être stockés afin d’assurer des données fonctionnelles de bonne qualité provenant des cardiomyocytes extraits).

Traditionnellement, l’évaluation des propriétés mécaniques myocardiques a été essayée la plupart du temps dans les préparations multicellulaires, telles que les muscles papillaires et le trabeculae1,2. Les bandes multicellulaires de muscles cardiaques incluent une population hétérogène de cellules, y compris les cardiomyocytes contractiles avec un modèle inconnu d’orientation et de génération de force, l’activité électrique et les distributions de stress/tension aussi bien qu’une matrice environnante de tissu conjonctif3,4. Une préparation sans collagène et contenant un seul cardiomyocyte permettrait de mesurer la longueur du sarcomère et les propriétés contractiles croisées d’une manière très précise etcontrôlée 5,6. Par conséquent, au cours des quatre dernières décennies, plusieurs méthodologies ont été développées permettant d’étudier les propriétés mécaniques, contractiles et de relaxation d’un seul cardiomyocyte6,7. La fonction contractile de ces cellules dépend fortement de la longueur du sarcomère et de la cinétique du cycle transmanie3. Ainsi, il est souhaitable d’étudier la fonction musculaire directement dans les cellules cardiaques isolées simples, considérant qu’il permet d’évaluer la longueur et la performance du sarcome ainsi que la fonction transversale et les propriétés contractiles. Cependant, isoler et attacher des cardiomyocytes fonctionnels avec une résolution optique raisonnable de sarcomere tout en enregistrant la mesure de force au niveau de μN est toujoursprovocant et évoluant 3,6. D’autres défis sont la logistique qui doit être installée pour isoler les cardiomyocytes des biopsies fraîchement recueillies. L’imprévisibilité de la collecte de biopsies humaines, par exemple, peut compromettre la faisabilité des expériences.
En outre, les préoccupations éthiques concernant le remplacement, la réduction et le raffinement de l’expérimentation animale pour les procédures scientifiques (principes des 3R) ont favorisé les changements d’étude au niveau cellulaire et tissulaire, de préférence dans les biopsies humaines, ou dans les échantillons d’animaux plus petits. En effet, un raffinement progressif des méthodologies d’évaluation in vitro de la fonction cardiaque à un plus petit niveau de complexité permet une bonne intégration des résultats à l’ensemble du corps et de les traduire au scénario clinique7. Au total, l’utilisation d’échantillons stockés à -80 °C pour extraire les cardiomyocytes peut être une alternative attrayante.
Le tissu myocardique est coupé en petits morceaux et homogénéisé avec un mortier et un pilon. Le résultat de cette homogénéisation est une suspension des cellules empaquetées et isolées écorchées avec des degrés variables de dommages sarcolemmal, dans lequel le myoplasme est exposé au milieu de bain et tous les composants cellulaires sont lavés. Des structures telles que les myofibriles qui sont plus loin de la sarcolemma sont préservées. Ainsi, le raccourcissement sarcomere et les propriétés fonctionnelles associées à l’appareil myofibrillar sont maintenus intacts et peuvent êtreenregistrés 8,9.
Le système de mesure de la force cardiomyocyte se compose d’un moteur électromagnétique, utilisé pour ajuster la longueur des cardiomyocytes, et d’un transducteur de force, qui mesure la contraction cardiomyocyte isométrique. Un cardiomyocyte perméabilisé ou écorché est placé dans une chambre expérimentale contenant une solution relaxante ([Ca2+] < 10 nM) et collée au silicium à 2 aiguilles minces : l’une attachée au moteur et l’autre au transducteur de force. Un système optique est utilisé pour déterminer la morphologie des cardiomyocytes et la longueur du sarcome. Le protocole expérimental consiste souvent en une série d’enregistrements de force sur des solutions tampons contenant différentes concentrations de Ca2+, la détermination de la cinétique entre actine et myosine et la mesure de la tension passive des cardiomyocytes montés à des longueurs de sarcomere prédéfinies (figure 1). L’isolement des cardiomyocytes perméabilisés des échantillons myocardiques congelés dans de l’azote liquide (et ensuite stockés à -80 °C) est une technique qui utilise la mécanique cellulaire et la biochimie protéique pour mesurer la force maximale ca2+activée (active) par zone transversale (Tactive,kN◊m-2),Ca2+-indépendante tension (passive) (Tpassive,kN◊m-2),myofilaments Ca2+-sensibilité (pCa50),monofilaments cooperativity (nHill), taux de réaménagement de la force (ktr) ainsi que dépendances de longueur sarcomere de Tactive,Tpassive, pCa50, nHill et ktr.
Le but de ce protocole est d’illustrer et de résumer le potentiel du système de mesure de la force cardiomyocyte comme une procédure fiable pour évaluer les propriétés mécaniques fonctionnelles des cardiomyocytes à peau unique isolés d’échantillons congelés de différentes espèces.

<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

Dans cet article, nous décrivons les étapes nécessaires pour isoler un seul cardiomyocyte permeabilisé (« écorché ») et l’attacher à un appareil de mesure de la force et un moteur pour effectuer des études fonctionnelles. Ces études permettront de mesurer la rigidité cardiomyocyte (force passive) et son activation avec différentes solutions contenant du calcium (Ca2+) pour déterminer, entre autres: le développement maximal de la force, le myofilament Ca2+-sensibilité (pCa50),la cooperativité (nHill) et le taux de réaménagement de la force (ktr). Cette méthode permet également de déterminer les effets des médicaments agissant directement sur les myofilaments et de l’expression de protéines recombinantes exogènes sur les propriétés actives et passives des cardiomyocytes. Cliniquement, les études écorchées de cardiomyocyte mettent en évidence la pathophysiologie de beaucoup de maladies myocardiques et permettent l’évaluation in vitro de l’impact des interventions thérapeutiques ciblant les myofilaments. Au total, cette technique permet de clarifier la pathophysiologie cardiaque en étudiant les corrélations entre les paramètres in vitro et in vivo dans les modèles animaux et les tissus humains obtenus lors d’une chirurgie à cœur ouvert ou d’une greffe.

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

Ce protocole vise à décrire étape par étape la technique d’extraction et d’évaluation de la fonction cardiaque à l’aide de cardiomyocytes écorchés. Cette méthodologie permet la mesure et la modulation aiguë de la fonction myofilament à l’aide de petites biopsies congelées qui peuvent être recueillies à différents endroits cardiaques, des souris aux hommes.

Évaluation in vitro de la fonction cardiaque à l’aide de cardiomyocytes écorchés

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 vues.  Universidade do Porto. Ce protocole vise à décrire étape par étape la technique d’extraction et d’évaluation de la fonction cardiaque à l’aide de cardiomyocytes écorchés. Cette méthodologie permet la mesure et la modulation aiguë de la fonction myofilament à l’aide de petites biopsies congelées qui peuvent être recueillies à différents endroits cardiaques, des souris aux hommes.

Vidéo: 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.

L&#39;évaluation de la fonction cardiaque et Energétique dans les coeurs isolés de souris à l&#39;aide<sup&gt; 31</sup&gt; P NMR Spectroscopy

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

Recherche

Médecine

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.

L&#39;évaluation de la fonction cardiaque et la morphologie du myocarde en utilisant de petits Inversion Recovery Look-casier animale (SALLI) IRM chez les 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 ...

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.

Évaluation de ventriculaire droite Structure et fonction dans le modèle de souris de l&#39;artère pulmonaire constriction par échocardiographie transthoracique

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.

Évaluation transcutanée de la fonction rénale dans Rongeurs Conscious

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.

préparation et<em&gt; In vitro</em&gt; Caractérisation des aimanté miR-modifié cellules endothéliales

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.

Évaluation de la fonction microvasculaire tissu adipeux humain en utilisant la vidéomicroscopie

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.

Normothermique Ex Situ coeur Perfusion en Mode de travail : évaluation de la fonction cardiaque et le métabolisme

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.

Évaluation échocardiographique de l'anatomie cardiaque et de la fonction chez les rats adultes

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.

Utilisation d’une biopsie chimique pour l’évaluation de la qualité de la greffe

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;

Méthode invasive semi-minimale pour induire l’infarctus du myocarde chez les rats et l’évaluation de la fonction cardiaque par un système cardiaque de travail isolé

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