Name: contractility measurements on isolated papillary muscles for the investigation of cardiac inotropy in mice
Uploaded: 2015-09-17T15:00:00
Description: Watch this Scientific Journal Video about Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice at JoVE.com

Dette arbejde blev støttet af Deutsche Forschungsgemeinschaft (KFO 196 &quot;Signaltransduktion bei adaptativen und maladaptiven kardialen Ombygning-Prozessen&quot;, FR 1638 / 1-2) og af DZHK (tysk center for Cardiovascular Research, en del af de tyske Centres of Health Research , som er en BMBF (tyske ministerium for uddannelse og forskning) initiativet).

BEMÆRK: De grundlæggende trin i isoleringsproceduren er vist i figur 1 Alle trin er beskrevet detaljeret i den følgende protokol.. Papillærmusklen isolation, montering i organbadet kammer, indsamling og analyse udføres i en fortløbende og obligatorisk tidshorisont. Alle dyreforsøg blev udført i overensstemmelse med tysk lovgivning om beskyttelse af dyr, og blev godkendt af den etiske Review Board for universitetet i Heidelberg. <p class="jove_tit...

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G., Jewell, B. R., Wood, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+the+contractility+of+mammalian+myocardium+at+low+rates+of+stimulation.">Studies of the contractility of mammalian myocardium at low rates of stimulation.</a> J Physiol. 254 (1), 1-17 (1976).</li><li>Pieske, B., Maier, L. S., Schmidt-Schweda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sarcoplasmic+reticulum+Ca2++load+in+human+heart+failure.">Sarcoplasmic reticulum Ca2+ load in human heart failure.</a> Basic Res Cardiol. 97, 163-171 (2002).</li><li>Koch-Weser, J., Blinks, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Influence+of+the+Interval+between+Beats+on+Myocardial+Contractility.">The Influence of the Interval between Beats on Myocardial Contractility.</a> Pharmacol Rev. 15, 601-652 (1963).</li><li>Bocalini, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+remodeling+after+large+infarcts+in+rat+converts+post+rest-potentiation+in+force+decay.">Myocardial remodeling after large infarcts in rat converts post rest-potentiation in force decay.</a> Arq Bras Cardiol. 98 (3), 243-251 (2012).</li><li>Juggi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ischemia-reperfusion+on+the+post-rest+inotropy+of+isolated+perfused+rat+heart.">Effect of ischemia-reperfusion on the post-rest inotropy of isolated perfused rat heart.</a> J Cell Mol Med. 6 (4), 621-630 (2002).</li><li>Lakatta, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+Bowditch:+the+convergence+of+cardiac+chronotropy+and+inotropy.">Beyond Bowditch: the convergence of cardiac chronotropy and inotropy.</a> Cell Calcium. 35 (6), 629-624 (2004).</li><li>Taylor, D. G., Parilak, L. D., LeWinter, M. M., Knot, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+the+rat+left+ventricle+force+and+Ca2++-frequency+relationships:+similarities+to+dog+and+human.">Quantification of the rat left ventricle force and Ca2+ -frequency relationships: similarities to dog and human.</a> Cardiovasc Res. 61 (1), 77-86 (2004).</li><li>Schmidt, U., Hajjar, R. J., Gwathmey, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+force-interval+relationship+in+human+myocardium.">The force-interval relationship in human myocardium.</a> J Card Fail. 1 (4), 311-321 (1995).</li><li>Rossman, E. I., Petre, R. E., Chaudhary, K. W., Piacentino, V. 3. r. d., Janssen, P. M., Gaughan, J. P., Houser, S. R., Margulies, K. 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I dette manuskript beskriver vi en fremgangsmåde til at undersøge kontraktilitet murine papillarmuskel in vitro, som kan bruges til at besvare en række videnskabelige spørgsmål i forbindelse med hjerte fysiologi og patologi hos mus samt at støtte analysen af transgene linier og opdagelsen af nye farmaceutiske metoder til behandling af hjerteproblemer dysfunktioner. Vi illustrerer anvendelsen af denne metode til at vurdere fysiologiske, patologiske og farmakologiske egenskaber af hjertemusklens kontraktili...

Undersøgelsen af ​​proteiner som ionkanaler henviser deres rolle for hjerte-kontraktilitet er afgørende for at opdage forskellige patomekanismer og fastsætte nye terapeutiske strategier for hjertesygdomme såsom iskæmi og hjertesvigt. Kontraktile funktion af mammale cardiomyocytter vides at blive moduleret af forskellige ionkanaler, transportører og andre proteiner. Potentiel handling fremkaldt aktivering af spænding afhængig sarcolemmal L-typen Ca 2 + kanaler fører til Ca2 + indstrømning fra ekstracellulære rum og senere til Ca 2 + induceret Ca 2+ frigivelse (CICR) 1, som udløser cellulære 2 sammentrækning. Ca 2+ -signaling spiller en central rolle i hjertets sammentrækningsevne og tilpasning til fysiologisk eller patologisk stress. Katekolaminer aktiverer hjerte- p-adrenerge receptorer og derved stimulere adenylylcyklase (AC), som syntetiserer cAMP. Aktiveres, Protein kinase A (PKA) phosphorylerer forskellige intracellulære og membran associerede proteiner ligesom L-type Ca 2 + kanaler, phospholamban og ryanodine receptorer resulterer i ændring af Ca 2 + transienter og hjerte-kontraktilitet 1,3,4. cAMP nedbrydes af phosphodiesterase (PDE). Aktivering af andre end p-adrenoceptorer Gs-koblede receptorer fører også til akkumulering af cAMP. Teknikken med kontraktilitet målinger i isolerede ventrikulære muskelstrimler er veletableret for større pattedyrarter 5-8. Baseret på muligheden for gen-targeting i mus er det vigtigt at etablere metoder til at analysere murine cardiac fysiologi. Men eksisterende data om de fysiologiske egenskaber af isolerede præparater muskel i mus varierer afhængigt af eksperimentelle betingelser 9-12. Den beskrevne metode anvendes til at analysere hjertets kontraktilitet af venstre ventrikels papillærmuskel prepræparater in vitro. Undersøgelse af hjertets kontraktilitet udføres i fravær af påvirkninger ændring hjertets kontraktilitet in vivo, ligesom blodtryk, neurohumoral stimulering og fysisk eller metabolisk stress. Den bankende sats for den ordregivende muskel præparat kan strengt defineres og ændres vilkårligt. Spjæt kraft kan analyseres i sammenhæng med specifikke stimuli såsom calcium koncentration, slå frekvens eller temperatur. Desuden kan denne fremgangsmåde anvendes til at undersøge forskellige signalvej komponenter, og at sammenligne hjertets funktion af genetisk modificerede musemodeller ved at styre eksperimentelle betingelser nævnt ovenfor.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5761&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>D9434&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P5280&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td align="right">223506</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate</td>
			<td>Sigma-Aldrich</td>
			<td align="right">230391</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>P 5655</td>
			<td></td>
		</tr>
		<tr>
			<td>2,3-Butanedione monoxime</td>
			<td>Sigma-Aldrich</td>
			<td>B0753</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma-Aldrich</td>
			<td>F3917</td>
			<td>Hazard statement H312, solve in DMSO</td>
		</tr>
		<tr>
			<td>3-?Isobutyl-?1-?methylxanthine</td>
			<td>Sigma-Aldrich</td>
			<td>I5879</td>
			<td>Hazard statement H 302, solve in DMSO</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoprenaline hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>I5627</td>
			<td>Hazard statement H 315-H319-H335</td>
		</tr>
		<tr>
			<td>Sodium Heparine 250.000 IE/10ml</td>
			<td>ratiopharm</td>
			<td>PZN 3874685</td>
			<td></td>
		</tr>
		<tr>
			<td>Histamine dihydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>H7250</td>
			<td>Hazard statement H 315-H 317-H319- H334-H335</td>
		</tr>
	</tbody>
</table>

contractility measurements on isolated papillary muscles for the investigation of cardiac inotropy in mice

Papillærmuskel isoleret fra voksne mus hjerter kan anvendes til at studere hjertets kontraktilitet under forskellige fysiologiske / patologiske tilstande. De kontraktile egenskaber kan vurderes uafhængigt af ydre påvirkninger såsom vaskulær tonus eller neurohumorale status. Det forestiller en videnskabelig tilgang mellem enkelt celle målinger med isolerede hjertemyocytter og in vivo studier som ekkokardiografi. Således papillærmuskel præparater tjene som en glimrende model til at studere hjertets fysiologi / patofysiologi og kan anvendes til undersøgelser som modulationen af ​​farmakologiske midler eller udforskning af transgene dyremodeller. Her beskriver vi en fremgangsmåde til isolering af murine venstre forreste papillærmuskel at undersøge hjertets kontraktilitet i et organbad setup. I modsætning til en muskelstrimlen præparat isoleret fra den ventrikulære væg, kan papillærmusklen fremstilles i toto uden at beskadige musklen tissue alvorligt. Organbadet setup består af flere temperaturkontrollerede, gasset og elektrodernes udstyret organbad kamre. Det isolerede papillarmuskel er fastgjort i organbadet kammeret og elektrisk stimuleret. Den fremkaldte twitch kraft registreres ved hjælp af en tryktransducer og parametre såsom spjæt kraftamplituden og spjæt kinetik analyseres. Forskellige forsøgsprotokoller kan udføres for at undersøge calcium- og frekvensafhængig kontraktilitet samt dosisresponskurver af kontraktile midler, såsom katekolaminer eller andre lægemidler. Derudover kan patologiske tilstande som akut iskæmi simuleres.

Protokollen for dette håndskrift for kontraktilitet målinger af isolerede murine papillære muskel præparater er indstillet til optimale betingelser for at opnå reproducerbare forsøgsresultater under fysiologiske betingelser. For at definere optimale eksperimentelle betingelser, vi udførte pilotforsøg varierende organbadet temperatur og ekstracellulær koncentration calcium (se også 12). Den her beskrevne protokol blev udført med et ekstracellulært calcium koncentration på 1,5 mM og en temperatur p...

Watch this Scientific Journal Video about Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice at JoVE.com

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Murint venstre ventrikel papillær muskel kan anvendes til at undersøge hjertets kontraktilitet in vitro. Denne artikel beskriver i detaljer isolation og forsøgsprotokoller til at studere hjerte-kontraktile egenskaber.

Kontraktilitet Målinger på isolerede papillærmuskler for undersøgelse af Cardiac inotropi i mus

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

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