Name: isolation of high quality murine atrial and ventricular myocytes for simultaneous measurements of ca2+ transients and l-type calcium current
Uploaded: 2020-11-03T15:00:00
Description: Watch this Scientific Journal Video about Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current at JoVE.com

Dette arbejde blev støttet af German Research Foundation (DFG; Klinikerforskerprogram i vaskulær medicin (PRIME), MA 2186/14-1 til P. Tomsits og D. Schüttler; VO1568/3-1, IRTG1816 og SFB1002 projekt A13 til N. Voigt), German Research Foundation under Tysklands Excellence Strategy (EXC 2067/1- 390729940 til N. Voigt), German Centre for Cardiovascular Research (DZHK; 81X2600255 til S. Clauss og N. Voigt; 81Z0600206 til S. Kääb), Corona Foundation (S199/10079/2019 til S. Clauss), ERA-NET on Cardiovascular Diseases (ERA-CVD; 01KL1910 til S. Clauss), Heinrich-og-Lotte-Mühlfenzl Stiftung (til S. Clauss) og Else-Kröner-Fresenius Foundation (EKFS 2016_A20 til N. Voigt). Bidragyderne havde ingen rolle i udarbejdelsen af manuskripter.

Alle dyreforsøg blev godkendt af Niedersachsen Animal Review Board (LAVES, AZ-18/2900) og blev udført i overensstemmelse med alle institutionelle, nationale og internationale retningslinjer for dyrevelfærd.
1. Forudgående aftaler
<ol><li>Der fremstilles 1 liter 10x perfusionsbuffer (tabel 1), 500 ml 1x perfusionsbuffer (tabel 2), 50 ml fordøjelsesbuffer (tabel 3), 10 ml stopbuffer (tabel 4), 1 liter tyrodeopløsning (tabel 5</st...

<ol>
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Denne artikel giver en nem og funktionel måde at opnå atriale og ventrikulære myocytter af høj kvalitet fra den samme mus til patch-clamp-undersøgelser med samtidige calciumtransiente optagelser. Kvaliteten af de opnåede data afhænger stærkt af kvaliteten af celleisoleringen. Som nævnt ovenfor er mange metoder til isolering af murine kardiomyocytter tidligere beskrevet 9,10,11,12. De i...

Hjertearytmier er almindelige og en af de nuværende store sundhedsudfordringer, da de påvirker millioner af mennesker verden over. Arytmier er forbundet med høj sygelighed og dødelighed 1,2 og repræsenterer den underliggende årsag til størstedelen af pludselige hjertedødsfald3. Opdaterede behandlingsmuligheder har forbedret patientens overlevelse, men er stadig hovedsageligt symptomatiske behandlinger snarere end at målrette de underliggende mekanismer. Disse behandlinger har således begrænset effekt og kan ofte forårsage alvorlige bivirkninger 4,5,6. En forbedring af de nuværende behandlingsmuligheder kræver indsigt i den underliggende patofysiologi, hvilket skaber behov for egnede modeller at studere. Små dyremodeller - og specifikt musemodeller - spiller en afgørende rolle i arytmiforskning, da de giver mulighed for at studere nøglemekanismer for arytmogenese, for eksempel den genetiske indvirkning på cellulær elektrofysiologi, ionkanalfunktion eller calciumhåndtering 7,8.
Til dette formål kræves isolerede atriale og ventrikulære kardiomyocytter af tilstrækkelig mængde og levedygtighed. Et bredt spektrum af forskellige isolationsmetoder til opnåelse af atriale og ventrikulære myocytter er tidligere blevet beskrevet 9,10,11,12,13, og nogle grupper har præsenteret data fra samtidige målinger af L-type strøm og calciumstrøm induceret calciumtransienter fra enten atriel 14 eller ventrikulær 15 murin kardiomyocytter. Men så vidt vi ved, er der ingen tilgængelige data om atriale såvel som ventrikulære målinger fra et dyr. Forskere fokuserer på en bred vifte af emner lige fra elektrofysiologi til proteomics, funktionelle undersøgelser som cellekontraktilitet eller proteininteraktioner, mitokondriefunktion eller genetik - alle med behov for isolerede kardiomyocytter. Mange af de offentliggjorte protokoller er således ikke specifikt udviklet til patch clamp studier, hvilket fører til begrænsede udbytter og utilstrækkelig cellekvalitet til patch clamp studies. Således kan samtidige patch clamp og calcium transiente målinger af atriale og ventrikulære celler isoleret fra et dyr ikke udføres med etablerede protokoller.
Isolering af murin - især atriale - myocytter til patch clamp eksperimenter forbliver udfordrende. Denne artikel giver en enkel og hurtig metode til isolering af højkvalitets murin atriale og ventrikulære myocytter via retrograd enzymbaseret Langendorff-perfusion, som efterfølgende muliggør samtidige målinger af både nettomembranstrøm og strøminducerede calciumtransienter fra et dyr. Denne artikel uddyber en protokol til isolering af atriale og ventrikulære myocytter afledt af vildtypemus og mus, der bærer genetiske mutationer. Denne protokol kan bruges til både han- og hunmus. Myocytisolationen, billederne og de repræsentative resultater, der er beskrevet nedenfor, blev opnået fra vildtype C57Bl/6-mus i en alder af 6 (± 1) måneder. Ikke desto mindre er denne protokol med succes blevet brugt til mus i forskellige aldre fra 2 til 24 måneder med forskellige genotyper. Figur 1 viser isolationsopsætningen og et nærbillede af et kanyleret hjerte under enzymperfusion.

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

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

Musemodeller spiller en afgørende rolle i arytmiforskning og gør det muligt at studere nøglemekanismer for arytmogenese, herunder ændret ionkanalfunktion og calciumhåndtering. Til dette formål er atriale eller ventrikulære kardiomyocytter af høj kvalitet nødvendige for at udføre patch-clamp-målinger eller for at undersøge calciumhåndteringsabnormiteter. Det begrænsede udbytte af kardiomyocytter af høj kvalitet opnået ved nuværende isolationsprotokoller tillader imidlertid ikke begge målinger i samme mus. Denne artikel beskriver en metode til at isolere højkvalitets murin atriale og ventrikulære myocytter via retrograd enzymbaseret Langendorff-perfusion til efterfølgende samtidige målinger af calciumtransienter og L-type calciumstrøm fra et dyr. Mushjerter opnås, og aorta kanyleres hurtigt for at fjerne blod. Hjerter perfuseres derefter først med en calciumfri opløsning (37 °C) for at dissociere vævet i niveauet af interkalerede skiver og derefter med en enzymopløsning indeholdende lidt calcium for at forstyrre ekstracellulær matrix (37 °C). Det fordøjede hjerte dissekeres efterfølgende i atria og ventrikler. Vævsprøver hakkes i små stykker og opløses ved omhyggelig pipettering op og ned. Den enzymatiske fordøjelse stoppes, og cellerne genindføres trinvist til fysiologiske calciumkoncentrationer. Efter belastning med en fluorescerende Ca2+-indikator fremstilles isolerede kardiomyocytter til samtidig måling af calciumstrømme og transienter. Derudover diskuteres isolationsfaldgruber, og patch-clamp-protokoller og repræsentative spor af L-type calciumstrømme med samtidige calciumtransiente målinger i atriale og ventrikulære murine myocytter isoleret som beskrevet ovenfor tilvejebringes.

Isolationsudbyttet bestemmes efter calciumgentilførsel ved pipettering af 10 μL cellesuspension på et mikroskopglas. Mere end 100 levedygtige, stavformede, ikke-kontraherende celler/10 μL til atriecelleisolering og mere end 1.000 levedygtige, stavformede, ikke-kontraherende celler/10 μL til ventrikulær celleisolering betragtes som tilstrækkeligt udbytte og opnås almindeligvis ved anvendelse af denne protokol. Atrieceller opnået med denne protokol viste en række forskellige celletyper indeholdende celler i hjert...

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

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

Musmodeller gør det muligt at studere nøglemekanismer for arytmogenese. Til dette formål er kardiomyocytter af høj kvalitet nødvendige for at udføre patch-clamp-målinger. Her beskrives en metode til isolering af murine atriale og ventrikulære myocytter via retrograd enzymbaseret Langendorff-perfusion, som muliggør samtidige målinger af calciumtransienter og L-type calciumstrøm.

Isolering af murin atriale og ventrikulære myocytter af høj kvalitet til samtidige målinger afCa2+ transienter og L-type calciumstrøm

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

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Biology

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

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

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

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

Isolering og Kv-optagelser i murine atrielle og ventrikulære cardiomyocytter

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

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

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

Isolering af Humane Atrielle Myocytter til samtidige målinger af Ca<sup&gt; 2 +</sup&gt; Transienter og membranstrømme

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

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

Cytosoliske Calcium Målinger i Nyrer epitelceller ved flowcytometri

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

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

Spænding og Calcium Dual Channel Optical Kortlægning af dyrkede HL-1 Atriel myocyt Monolayer

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte ...

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

Isolering af mitokondrier fra minimale mængder af Mouse Skeletmuskel for High Throughput Microplate Respiratory Målinger

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

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

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

Udvikling og karakterisering af<em&gt; In vitro</em&gt; Mikrokar Netværk og kvantitative målinger af endothelial [Ca<sup&gt; 2+</sup&gt;]<sub&gt; jeg</sub&gt; Og nitrogenoxid Produktion

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

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Metoder til isolering, Kultur og funktionel karakterisering af sinoatrialt Node Myocytter fra voksne mus

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

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

Samtidig måling af Mitokondriel Calcium og mitokondriemembranen Potentiale i levende celler ved Fluorescent Microscopy

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

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

Samtidig isolering af cardiomyocytter af høj kvalitet, endotelceller og fibroblaster fra en voksen rottehjerte

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