Name: assessment of sarcoplasmic reticulum calcium reserve and intracellular diastolic calcium removal in isolated ventricular cardiomyocytes
Uploaded: 2017-09-18T13:00:00
Description: Watch this Scientific Journal Video about Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes at JoVE.com

Dette arbejde blev støttet af tilskud fra National Natural Science Foundation of China (nr. 81100159, Dongwu Lai, 81401147, Juhong Zhang), medicinske og sundhed Science Program af Zhejiang provinsen (nr. 201646246, Dongwu Lai) og den Sundhedsvidenskab og Teknologi Plan af Hangzhou City (No. 2013A28, Ding Lin).

alle dyreforsøg blev udført efter protokoller godkendt af institutionelle Animal Care og brug Udvalget på eksperimentelle Research Center, Kina kinesisk medicinsk akademi og Zhejiang University.
 1. løsning forberedelse <ol><li>forberede alle løsninger, som beskrevet i tabel 1.</li></ol>
 2. isolation af venstre ventrikel (LV) Cardiomyocytes NOTE: LV cardiomyocytes er isoleret enzymatisk ved hjælp af en Langendroff pe...

<ol>
	<li>Capel, R. A., Terrar, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+Ca+(2+)-dependent+mechanisms+for+the+initiation+of+the+heartbeat.">The importance of Ca (2+)-dependent mechanisms for the initiation of the heartbeat.</a> Front Physiol. 6, 80 (2015).</li><li>Bers, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+cycling+and+signaling+in+cardiac+myocytes.">Calcium cycling and signaling in cardiac myocytes.</a> Annu Rev Physiol. 70, 23-49 (2008).</li><li>Zhao, S. M., Wang, Y. L., Guo, C. Y., Chen, J. L., Wu, Y. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+decay+of+Ca2++homeostasis+in+the+development+of+diabetic+cardiomyopathy.">Progressive decay of Ca2+ homeostasis in the development of diabetic cardiomyopathy.</a> Cardiovasc Diabetol. 13, 75 (2014).</li><li>Pereira, L., 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=Calcium+signaling+in+diabetic+cardiomyocytes.">Calcium signaling in diabetic cardiomyocytes.</a> Cell calcium. 56 (5), 372-380 (2014).</li><li>Coppini, 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=Late+sodium+current+inhibition+reverses+electromechanical+dysfunction+in+human+hypertrophic+cardiomyopathy.">Late sodium current inhibition reverses electromechanical dysfunction in human hypertrophic cardiomyopathy.</a> Circulation. 127 (5), 575-584 (2013).</li><li>Ferrantini, 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=R4496C+RyR2+mutation+impairs+atrial+and+ventricular+contractility.">R4496C RyR2 mutation impairs atrial and ventricular contractility.</a> J Gen Physiol. 147 (1), 39-52 (2016).</li><li>Li, L., Chu, G., Kranias, E. G., Bers, D. 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+myocyte+calcium+transport+in+phospholamban+knockout+mouse+relaxation+and+endogenous+CaMKII+effects.">Cardiac myocyte calcium transport in phospholamban knockout mouse relaxation and endogenous CaMKII effects.</a> Am J Physiol. 274, H1335-H1347 (1998).</li><li>Cheng, 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=CaMKII+inhibition+in+heart+failure,+beneficial,+harmful,+or+both.">CaMKII inhibition in heart failure, beneficial, harmful, or both.</a> Am J Physiol Heart Circ Physiol. 302, H1454-H1465 (2012).</li><li>Lai, 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=The+Rho+kinase+inhibitor,+fasudil,+ameliorates+diabetes-induced+cardiac+dysfunction+by+improving+calcium+clearance+and+actin+remodeling.">The Rho kinase inhibitor, fasudil, ameliorates diabetes-induced cardiac dysfunction by improving calcium clearance and actin remodeling.</a> J Mol Med (Berl). 95 (2), 155-165 (2017).</li><li>Lai, 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=Stretch+Current-Induced+Abnormal+Impulses+in+CaMKII&#948;+Knockout+Mouse+Ventricular+Myocytes.">Stretch Current-Induced Abnormal Impulses in CaMKII&#948; Knockout Mouse Ventricular Myocytes.</a> J Cardiovasc Electrophysiol. 24 (4), 457-463 (2013).</li><li>Xu, L., 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+of+L-type+calcium+current+and+cardiac+function+in+CaMKII{delta}+knockout+mice.">Alterations of L-type calcium current and cardiac function in CaMKII{delta} knockout mice.</a> Circ Res. 107 (3), 398-407 (2010).</li><li>Roussel, 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=Palmitoyl-carnitine+increases+RyR2+oxidation+and+sarcoplasmic+reticulum+Ca2++leak+in+cardiomyocytes:+Role+of+adenine+nucleotide+translocase.">Palmitoyl-carnitine increases RyR2 oxidation and sarcoplasmic reticulum Ca2+ leak in cardiomyocytes: Role of adenine nucleotide translocase.</a> Biochim Biophys Acta. 1852 (5), 749-758 (2015).</li><li>Yaras, 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=Effects+of+diabetes+on+ryanodine+receptor+Ca2++release+channel+(RyR2)+and+Ca2++homeostasis+in+rat+heart.">Effects of diabetes on ryanodine receptor Ca2+ release channel (RyR2) and Ca2+ homeostasis in rat heart.</a> Diabetes. 54 (11), 3082-3088 (2005).</li><li>Hu, Y., 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=Adenovirus-Mediated+Overexpression+of+O-GlcNAcase+Improves+Contractile+Function+in+the+Diabetic+Heart.">Adenovirus-Mediated Overexpression of O-GlcNAcase Improves Contractile Function in the Diabetic Heart.</a> Circ Res. 96 (9), 1006-1013 (2005).</li></ol>

Calcium flux frigivet fra SR er den største Ca2 + kilde for systole i hjertet. Til nogle omfang, amplitude af SR Ca2 + indhold og fraktioneret Ca afspejler2 + frigivet fra SR den SR Ca2 + reserve tilgængelige for hjertets sammentrækning. På den anden side afhænger Ca2 + reserven af SR af SR Ca2 + reuptake, Ca2 + lækage af SR og deres balance evne på tværs af SR under diastolen12,13</su...

Intracellulære calcium ([Ca2 +]jeg) genanvendelse spiller en afgørende rolle i reguleringen af systolisk og diastolisk funktion i cardiomyocytes1. Som vi ved, indleder calcium-induceret Ca2 + release excitation-sammentrækning kobling, hvilket oversætte det elektriske signal til sammentrækning. Membran depolarisering aktiverer sarcolemmal L-type Ca2 + kanaler, som inducerer Ca2 + udledning fra SR i cytoplasma via ryanodine receptorer 2 (RyR2). Den forbigående forhøjede cytoplasmatisk Ca2 + indleder sammentrækning af myofibrils. Under diastolen, cytoplasmatisk Ca2 + er reuptaken i SR med SR Ca2 +-ATPase 2 (SERCA2) og pumpes ud af cardiomyocyte via Na+-Ca2 + exchanger (NCX)2. Denne proces fører til sammentrækning-lempelse genbrug i cardiomyocyte.
Den kardiale SR er en intracellulær membran netværk, der omgiver de kontraktile maskineri. Det tjener som en Ca2 + reservoir for sammentrækning, og det reabsorbs intracellulære Ca2 + under afslapning. SR Ca2 + reserve til beats er praecise for hjertets kontraktilitet. I mellemtiden, fjernelse af intracellulære Ca2 + er kritisk for kardiale diastolen. Nogle patofysiologiske betingelser såsom diabetes og hjertesvigt, nedsat Ca2 + clearance og deprimeret SR Ca2 + kan butik i cardiomyocytes være involveret i processen med hjerte dysfunktion2,3 ,4.
Til måling af SR Ca2 + release og diastolisk Ca2 + fjernelse i cardiomyocytes, der er to udbredte tilgange: integriteten af NCX nuværende baseret på patch-klemme5,6, og koffein-induceret Ca 2 + pulse baseret på Ca2 + fluorescens imaging7,8,9. Den tidligere tilgang afhænger det faktum, at den Ca2 + frigivet fra SR er i vid udstrækning pumpes ud af cellen af NCX. Dog, denne fremgangsmåde er begrænset af sine krav af avanceret udstyr og dygtige drift. I den foreliggende undersøgelse beskriver vi en praktisk tilgang til at vurdere SR Ca2 + reserve og Ca2 + fjernelse i myocytes ved at måle en koffein-induceret Ca2 + pulse baseret på en Ca2 + fluorescens imaging system. Kort, intracellulære Ca2 + Fluorescens er indiceret ved Fura-2. Ved en sammenknytning af stimulation system og perfusion, præsenterer vi et program for skifter perfusion og pacing system automatisk. 10 mM koffein var ansat til hurtigt inducerer samlede Ca2 + udgivelse i SR. Eksponentiel henfald tid konstanter (Tau) af calcium transienter og koffein-induceret calcium pulser blev indhentet fra mono-eksponentiel kurve montering, som afspejler SERCA og NCX bidrag til diastolisk Ca2 + fjernelse i overensstemmelse hermed.

<table>
	<tbody>
		<tr>
			<td>NaCl</td>
			<td>Alfa Aesar</td>
			<td>E31K43</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Alfa Aesar</td>
			<td>I02T070</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Alfa Aesar</td>
			<td>G22u018</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPEs</td>
			<td>Sigma</td>
			<td>SLBM 7880V</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Alfa Aesar</td>
			<td>10189341</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Alfa Aesar</td>
			<td>10154048</td>
			<td></td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Alfa Aesar</td>
			<td>10144B17</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Alfa Aesar</td>
			<td>F21S033</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Alfa Aesar</td>
			<td>C31U038</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamic</td>
			<td>Alfa Aesar</td>
			<td>10149849</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Alfa Aesar</td>
			<td>J5407a</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>SLBM6826V</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase A</td>
			<td>Roche</td>
			<td>10103586001</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type II</td>
			<td>Worthington</td>
			<td>45k16005</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Roche</td>
			<td>735094</td>
			<td></td>
		</tr>
		<tr>
			<td>caffeine</td>
			<td>Sigma</td>
			<td>C0750</td>
			<td></td>
		</tr>
		<tr>
			<td>Fura-2 AM</td>
			<td>Invitrogen</td>
			<td>F1201</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>Olympus IX 71</td>
			<td></td>
		</tr>
		<tr>
			<td>Langendorff system</td>
			<td>Beijing Syutime Technology Co</td>
			<td>PlexiThermo-S-LANGC</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Marchauser</td>
			<td>MM33 links</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion chamber</td>
			<td>IonOptix</td>
			<td>FHD</td>
			<td></td>
		</tr>
		<tr>
			<td>Valve Controlled Gravity Perfusion System</td>
			<td>ALA</td>
			<td>VC 3-8</td>
			<td></td>
		</tr>
		<tr>
			<td>valve commander software</td>
			<td>ALA</td>
			<td>VC 3 1.0.1.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision flow regulator</td>
			<td>Delta Med</td>
			<td>3204315</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-Barrel Manifold Perfusion Pencil</td>
			<td>AutoMate Scientific</td>
			<td>04-08-[360]</td>
			<td></td>
		</tr>
		<tr>
			<td>Micron Removable Tip</td>
			<td>AutoMate Scientific</td>
			<td>360um i.d.</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence Measurement and Cell Dimensioning Systems</td>
			<td>IonOptix</td>
			<td>Hyperswitch</td>
			<td></td>
		</tr>
		<tr>
			<td>Recording software</td>
			<td>IonOptix</td>
			<td>IonWizard 6.2.59</td>
			<td></td>
		</tr>
		<tr>
			<td>Stimulator</td>
			<td>IonOptix</td>
			<td>MyoPacer EP</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague-dawley rats</td>
			<td>Beijing Vital River Laboratory Animal Technology Co.</td>
			<td>SCXK 2016-01-007436</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of sarcoplasmic reticulum calcium reserve and intracellular diastolic calcium removal in isolated ventricular cardiomyocytes

Intracellulære calcium genanvendelse spiller en afgørende rolle i reguleringen af systolisk og diastolisk funktion i cardiomyocytes. Hjerte sarcoplasmic reticulum (SR) fungerer som en Ca2 + reservoir for sammentrækning, hvilke reuptakes intracellulære Ca2 + under afslapning. SR Ca2 + reserve til beats er praecise for hjerte contractibility, og fjernelse af intracellulære Ca2 + er kritisk for diastolisk hjertefunktion. Under nogle patofysiologiske tilstande, såsom diabetes og hjertesvigt, kan nedsat calcium clearance og SR Ca2 + butik i cardiomyocytes inddrages i udviklingen af cardiac dysfunktion. Her, beskriver vi en protokol for at vurdere SRCa2 + reserve og diastolisk Ca2 + fjernelse. Kort, et enkelt cardiomyocyte blev enzymatisk isoleret, og den intracellulære Ca2 + fluorescens drejehastigheden Fura-2 blev optaget af et calcium imaging system. For at ansætte koffein for inducerende samlede SR Ca2 + release, forudindstillede vi en automatisk perfusion switchprogram af interlinking-mekanismen stimulation- og perfusion-systemet. Derefter, mono-eksponentialkurve montering blev brugt til at analysere henfald tid konstanter af calcium transienter og koffein-induceret calcium pulser. I overensstemmelse hermed, bidrag fra SR Ca2 +-ATPase (SERCA) og Na+-Ca2 + exchanger (NCX) til diastolisk calcium fjernelse blev evalueret.

Her, vi illustrere streptozotocin (STZ) - induceret diabetisk rotter (DM) og alder-matchede Sprague - Dawley (SD) rotter f.eks. 8-uge-forhenværende mandlige SD rotter (200 ± 20 g) modtaget en enkelt intraperitoneal injektion af STZ (70 mg/kg, ip) til DM gruppe eller citrat buffer for kontrolgruppen. Én uge efter administration af STZ rotter med blodglukose &#62; 16,7 mmol/L blev anset for diabetisk. Efter 8 uger blev LV myocytes enzymatisk isoleret. Efter calcium genindsætning er der ...

Watch this Scientific Journal Video about Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes at JoVE.com

Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes

Intracellulære calcium genanvendelse spiller en afgørende rolle i reguleringen af systolisk og diastolisk funktion i cardiomyocytes. Her, beskriver vi en protokol for at vurdere sarcoplasmic reticulum Ca2 + reserve og diastolisk calcium fjernelse funktion i cardiomyocytes af en calcium imaging system.

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Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum ...

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