Diese Arbeit wurde durch Fördermittel der Deutschen Forschungsgemeinschaft (DFG), Fritz-Thyssen-Stiftung und der Charité / MDC finanziert.

Ein. Tierische Anästhesie und Organentnahmen <ol><li> Betäuben die Maus durch intraperitoneale (ip) Injektion von Ketamin (200 mg / kg KG) und Xylazin (20 mg / kg KG). </li><li> Um anticoagulate injizieren 250 IU Heparin ip die Blutgerinnung und Thrombusbildung zu vermeiden. </li><li> Warten Sie, bis tiefe Narkose erreicht ist, die durch Areflexie gekennzeichnet. Um Areflexie, Test Cornealreflex durch leichtes Berühren der Hornhaut oder Testflug Reflex Schwanz kneifen überprüfen. </li><li> Übertragen Sie die Maus auf OP-Tisch und fixieren Sie sie in Rückenlage. </li><li> Inzision der Haut und der Bauchwand unterhalb der xiphoid und führen Clamshell Thorakotomie: Erweitern Sie den Schnitt auf beiden Seiten entlang der Rippenbogen und anschließend geschnitten Rippen in der medialen Axillarlinie, lenken die Brustkorb nach oben. </li><li> Öffnen Herzbeutel, finden großen Gefäße. Drücken Sie vorsichtig Herzen caudal zur besseren Darstellung der Aorta. Klemmen Sie die Aorta mit einer Pinzette. </li><li> Legen des Herzens in Konkavität eines Paars von n<html> cissors und sezieren alle verbindenden Gefäße mit einem einzigen Schnitt. Achten Sie darauf, um einen ausreichend großen Teil der aufsteigenden Aorta für Langendorff Kanülierung bewahren. </li><li> Übertragen herausgeschnitten Herz sofort in eine Petrischale mit eiskaltem und Pre-Oxygenwasser Lösung 1 (für Lösungen, siehe Tabelle 1) gefüllt. </li></ol> 2. Vorbereitung der Herz-und Langendorff Perfusion <ol><li> Kanülieren die Aorta mit einem 1.8F Stahlkanüle. Achten Sie darauf, Luftembolie zu vermeiden. </li><li> Fixieren Aorta auf der Kanüle mit einer chirurgischen Naht und bündig Koronarien mit 1 ml der Lösung 1. </li><li> Verbinden Kanüle mit einem Langendorff-Apparatur. </li><li> Stellen Sie sicher Zeit von Langendorff nach Thorakotomie Kanülisier nicht übersteigt 120 Sek. erweiterten Ischämie / Reperfusionsverletzung des Myokards zu verhindern. </li><li> Perfuse Herz mit 10 ml Ca 2 + kostenlose Lösung 2 (4 ml / min). </li><li> Perfuse Herz mit Collagenaselösung 3 für 8 min (4 ml / min). </li></ol>le &quot;&gt; 3. Mikrochirurgische Dissoziation von Cardiac Chambers <ol><li> Transfer Herz in einer vorgewärmten 100-mm Petrischale mit niedrigen Ca 2 +-Lösung 4. </li><li> Entfernen Sie vorsichtig Aorten-und andere nicht-Herzgewebe mit Schere und entsorgen Sie sie. </li><li> Separate Vorhöfe und Kammern und fahren Sie mit jeder Kammer getrennt. Halten Sie Zellen in Lösung 4 eingetaucht. Verwenden Sie kleine Mengen (weniger als 5 ml). </li></ol> 4. Weitere Dissoziation von Kardiomyozyten <ol><li> Atrial Kardiomyozyten: <ol><li> Zu individualisieren Vorhof Kardiomyozyten übertragen die Vorhöfe in einem separaten vorgewärmten 100-mm Kulturschale und distanzieren das Gewebe durch die sanft Auseinanderziehen mit feinen Pinzetten. Gewährleisten eine fast vollständige Dissoziation des Gewebes. </li><li> Verwenden Sie eine 1 ml-Pipette mit einer erweiterten Feuer-poliert mit Kunststoff Pipettenspitze, um die Zellen in 1 ml Lösung 5 auszusetzen für 5 min. </li><li> Trennen der Zellen von Schutt durch Verwendung eines Zellfilter (200 um Maschenweite). </li> &lt;li&gt; 5 ml Lösung 5 der Zellsuspension und Zentrifuge für 2 min bei 16 xg bei Raumtemperatur. </li><li> Die folgenden Schritte werden unter einer Zellkultur Haube betrieben. Überstand verwerfen und resuspendiert das Pellet in 5 ml Lösung 6. </li><li> Nach Sedimentation durch die Schwerkraft für 10 min in einem 15 ml-Röhrchen, für 1 min zentrifugieren bei 16 × g bei Raumtemperatur. Entfernen Sie den Überstand. Resuspendieren die Zellen je nach ihrer Menge in 1-5 ml Lösung 6. </li></ol></li><li> Ventrikuläre Kardiomyocyten: <ol><li> Sezieren die linksventrikuläre interessierenden Bereich mit feinen Pinzetten in 5 ml Lösung 4. </li><li> Auszusetzen Zellen durch sanftes Pipettieren, bis die meisten der Zellen getrennt sind. Übertragen der Zelle Lösung nach der Filterung (200 um Maschenweite) in einen 50-ml-Röhrchen, einen Volumen von 25 ml aufgefüllt. </li><li> Säule für 2 min bei 16 xg bei Raumtemperatur. </li><li> Die folgenden Schritte werden unter einer Zellkultur Haube betrieben. Überstand abnehmen, resuspendieren das Pellet in 25 mlLösung 6 und damit die Sedimentation der Zellen für 10 min. </li><li> Zählen Sie die Zellen und den Überstand, fügen Sie 25 bis 50 ml Lösung 6 auf die Zellen. </li></ol></li></ol> 5. Vorbereitung der Kardiomyozyten für Zelluläre Elektrophysiologie, biochemische oder IF Studies <ol><li> Für elektrophysiologischen Studien halten die Myozyten in Lösung 6 in einem 50 ml Röhrchen und hemmen Sedimentation. </li><li> Für biochemische Untersuchungen, z. B. Calcium imaging, Platte Myozyten auf Laminin beschichteten Zellkulturschale (Endkonzentration 20 ug / ml Laminin in PBS). </li><li> Für Immunfluoreszenzfärbung bereiten eine Zellkulturschale Platte mit Glasdeckgläschen und beschichtet mit Laminin-Lösung (Endkonzentration 50 ug / ml Laminin in PBS). <ol><li> Entfernen Sie die Lösung vor dem Beschichten Myozyten. Platte der Zellen und steuern die Zelldichte mit Hilfe eines Mikroskops. </li><li> Lassen Sie die Myozyten haften Slip für 1 Stunde bei 37 ° C in 2% CO 2 abzudecken, entfernen Sie die Lösungund beginnen sofort mit einer Standard-Färbeverfahren Protokoll. </li><li> Inkubieren mit Fixiermittel, beispielsweise 4% PFA in PBS (pH 7,5) für 10 min bei Raumtemperatur und folgen mit drei Waschschritten PBS für jeweils 5 min. </li><li> Um die Zellen zu hemmen und permeabilisieren unspezifische Antikörperbindung inkubieren Myocyten mit 10% Serum, 0,3% Triton, 0,2% BSA in PBS für 30 min bei Raumtemperatur. </li><li> Inkubieren mit dem primären Antikörper für 1 Std. bei 37 ° C und Waschen wie zuvor beschrieben. </li><li> Inkubieren mit dem sekundären Antikörper für 1 Std. bei Raumtemperatur. Um die Kerne Gegenfärbung und α-actinin Verwendung DAPI und Fluorochrom Phalloidin (Alexa Fluor 488, Invitrogen). </li><li> Nach dem Waschen, übertragen die Glasabdeckung rutscht vorsichtig auf Silan behandelten Objektträger und embed Zellen in Fluoreszenz-Eindeckmedium. </li></ol></li></ol> 6. Cellular Elektrophysiologie <ol><li> Führen Whole-Cell-Patch-Clamp-Aufnahmen auf freshly isoliert atrialen und ventrikulären Kardiomyocyten bei Raumtemperatur. </li><li> Übertragen erscheint gesund Kardiomyozyten in Perfusionskammer mit einem definierten Volumen extrazellulären Badlösung gefüllt. 117 mM NaCl, 4 mM KCl, 1 mM KH 2 PO 4, 4 mM NaHCO 3, 1,7 mM MgCl 2, 3 mM CoCl 2, 10 mM HEPES, 10 mM Glucose und 0,02 mM Tetrodotoxin (TTX), (pH 7,4) . Verwenden NaOH zur pH-Wert-Einstellung. </li><li> Mit geeigneten Patch Clamp Technik (dh IX71 inversen Mikroskop, ein Multiclamp 700B Verstärker, ein Erfassungssystem Digidata 1440A und PC mit pClamp10.3 Software (Molecular Devices)). </li><li> Verwenden Patch-Pipetten mit Widerständen von 3-5 MOhm, wenn mit intrazellulären Lösung, enthaltend 130 mM KCl, 2 mM MgCl 2, 11 mM HEPES, 11 mM EGTA, 5 mM Na 2 ATP, 0,4 mM Na 2 GTP, 5 mM Na 2 CP gefüllten und 4,9 mM CaCl 2 (pH 7,2). Verwenden KOH für pH-Wert-Einstellung. </li><li> Evoke außen K + Ströme duRing 4.5-sec Spannungsschritte zu testen Potentiale zwischen -60 und +50 mV in 10 mV-Schritten von einem Haltepotential von -70 mV nach einem 20-msek bis -40 mV Vorpuls. </li><li> Stellen Sie sicher, Leckströme sind immer &lt;100 pA. </li><li> Zum Sezieren von verschiedenen K +-Ströme verwenden spezifische Inhibitoren, wie 4-Aminopyridin (4-AP; ICN Biomedicals, Irvine, CA, USA), Heteropodatoxin 2 (HpTx2; Alomone) oder Tetraethylammonium (TEA, Sigma). Stammlösungen sollen in extrazellulären Badlösung hergestellt werden, und direkt auf eine möglichst enge Nachbarschaft der Zelle über eine Mikrospitze nach &quot;Baseline&quot; Aufnahmen. Äquilibrierung sollte für 2-3 min vor &quot;Droge&quot; Aufzeichnungen zugelassen werden. </li><li> Für die Analyse normalisieren Stromamplituden in einzelnen Zellen der Zellgröße (whole-cell Membran Kapazität). Analysieren von Daten mithilfe pClamp10.3 Software (Molecular Devices) oder vergleichbare Software offline. </li></ol>

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Wir haben nichts zu offenbaren.

Mit der wachsenden Entwicklung von gentechnisch veränderten Mauslinien die Herzfunktion und kardiale Pathologie zu Gen-Deletion zu studieren gibt es auch ein zunehmendes Interesse an spezialisierte Methoden, um Effekte der speziellen Gen-Deletion in vitro zu untersuchen. In unserem Labor untersuchen wir die Rolle einer Familie von Kv-Kanal zusätzlichen Untereinheiten auf die kardiale Repolarisation. Die KCNE Gene umfassen eine Familie von 5 Gene (KCNE1-5), die eine wichtige Rolle in der mens...

<table border="1"><tbody><tr><td> Name des Reagenz </td><td> Firma </td></tr><tr><td> Tetrodotoxin </td><td> Alomone </td></tr><tr><td> 4-Aminopyridin </td><td> ICN Biomedicals </td></tr><tr><td> Heteropodatoxin 2 </td><td> Alomone </td></tr><tr><td> Tetraethylammonium </td><td> Sigma Chemicals </td></tr><tr><td> Collagenase Typ 2 </td><td> Worthington </td></tr></tbody></table>

isolation and kv channel recordings in murine atrial and ventricular cardiomyocytes

KCNE Gene für eine kleine Familie von Kv-Kanal zusätzlichen Untereinheiten, die heteromeren Komplexe mit Kv-Kanal alpha-Untereinheiten bilden, um ihre funktionellen Eigenschaften zu modifizieren kodieren. Mutationen in KCNE Gene bei Patienten mit Herzrhythmusstörungen wie das Long-QT-Syndrom und / oder Vorhofflimmern gefunden. Allerdings bleibt die genaue molekulare Pathophysiologie, die zu diesen Krankheiten führt unklar. In früheren Studien die elektrophysiologischen Eigenschaften der Krankheit Mutationen in diesen Genen haben meist in heterologen Expressionssystemen untersucht worden und wir können nicht sicher sein, ob die berichteten Effekte können direkt in nativen Kardiomyozyten übersetzt werden. In unserem Labor setzen wir daher einen anderen Ansatz. Wir direkt zu untersuchen die Auswirkungen von KCNE Deletion in isolierten Herzmuskelzellen aus Knockout-Mäusen durch zelluläre Elektrophysiologie - eine einzigartige Technik, die wir beschreiben, in dieser Ausgabe des Journal of Visualized Experiments. Die Herzen von genetically entwickelt KCNE Mäusen schnell ausgeschnitten und montiert auf einem Langendorff-Apparatur durch Aorten Kanülierung. Freiem Ca 2 + in dem Myokard durch EGTA gebunden und Dissoziation von Herzmuskelzellen wird dann durch retrograde Perfusion der Koronararterien mit einem spezialisierten niedrigen Ca 2 +-Puffer, der Kollagenase erreicht. Atria, freien rechtsventrikulären Wand und die linke Herzkammer kann dann durch mikrochirurgische Techniken getrennt werden. Calcium wird dann langsam wieder auf isolierte Herzmuskelzellen in einem mehrstufigen Waschvorgangs zugegeben umfassend. Atrialen und ventrikulären Kardiomyozyten gesundes Aussehen ohne spontane Kontraktionen werden dann sofort an elektrophysiologische Untersuchungen durch Patch-Clamp-Technik oder andere biochemische Analysen innerhalb der ersten 6 Stunden nach der Isolierung unterzogen.

Isolierung von adulten murinen Kardiomyozyten aus gentechnisch veränderten Mäusen, die Funktion bestimmter Gene von Interesse in vitro-Studie hat sich zu einem mächtigen Werkzeug, um weiter zu verstehen kardialen Pathophysiologie. Dieses Verfahren wird derzeit nur von einer kleinen, aber wachsenden Zahl von Grundlagenforschung Labors weltweit verwendet. Allerdings können Isolierung von adulten ventrikulären murine Kardiomyozyten tückisch sein und muss gründlich und wiederholt von erfahrenen Händen durch...

Watch this Scientific Journal Video about Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes at JoVE.com

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

Kv-Kanal Dysfunktion ist mit Herzrhythmusstörungen verbunden. Um zu untersuchen, die molekularen Mechanismen, die zu solchen Arrhythmien nutzen wir eine systematische Protokoll zur Isolierung von atrialen und ventrikulären Kardiomyozyten aus Kv Kanal Nebenleistungen Untereinheit Knockout-Mäusen führen. Isolierte Herzmuskelzellen kann dann sofort für zellulare elektrophysiologische Studien, biochemischen oder Immunfluoreszenz (IF)-Assays verwendet werden.

Isolierung und Kv Kanal Recordings in Murine atriale und ventrikuläre Kardiomyozyten

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

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13.3K Aufrufe.  Charité Medical Faculty and Max-Delbrück Center for Molecular Medicine (MDC). Kv-Kanal Dysfunktion ist mit Herzrhythmusstörungen verbunden. Um zu untersuchen, die molekularen Mechanismen, die zu solchen Arrhythmien nutzen wir eine systematische Protokoll zur Isolierung von atrialen und ventrikulären Kardiomyozyten aus Kv Kanal Nebenleistungen Untereinheit Knockout-Mäusen führen. Isolierte Herzmuskelzellen kann dann sofort für zellulare elektrophysiologische Studien, biochemischen oder Immunfluoreszenz (IF)-Assays verwendet werden.

Serie: Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

Phänotypische Analyse und Isolierung von murinen hämatopoetischen Stammzellen und Lineage-festgelegte Vorläuferzellen

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Forschung

Biologie

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

Isolation und Kultur der Neugeborenen-Maus-Kardiomyozyten

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

Rekonstitution eines Kv-Kanal in Lipidmembranen für Strukturelle und Funktionelle Studien

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

Isolation und physiologische Analyse von Maus Kardiomyozyten

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

Isolation und Kultur der erwachsenen Maus Kardiomyozyten für Cell Signaling und<em&gt; In-vitro-</em&gt; Herzhypertrophie

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Ein-Kanal-Cell-Attached-Patch-Clamp-Aufnahme

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

Funktionelle Rekonstitution und Kanalaktivitätsmessungen von gereinigtem Wildtyp und mutierten CFTR-Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte 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 monolayers. HL-1 cells have been widely used as a 2-Dimensional cellular model for studying diverse aspects of cardiac physiology. However, it has been a great challenge to optically map calcium (Ca) transients and action potentials simultaneously from the same field of view in a cultured HL-1 atrial cell monolayer. This is because special handling and care is required to prepare healthy cells that can be electrically captured and optically mapped. Therefore, we have developed an optimal working protocol for dual channel optical mapping. In this manuscript, we have described in detail how to perform the dual channel optical mapping experiment. This protocol is a useful tool to enhance the understanding of action potential propagation and Ca kinetics in arrhythmia development.

This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

Spannung und Calcium Dual Channel Optical Mapping von Cultured HL-1 Vorhofmuskelzellen 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, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

Isolierung, Kultur und Transduction von erwachsenen Maus Kardiomyozyten

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

Subtype-specific Optical Action Potential Recordings in Human Induced Pluripotent Stem Cell-derived Ventricular Cardiomyocytes

Cardiomyocytes generated from human induced pluripotent stem cells (iPSC-CMs) are an emerging tool in cardiovascular research. Rather than being a homogenous population of cells, the iPSC-CMs generated by current differentiation protocols represent a mixture of cells with ventricular-, atrial-, and nodal-like phenotypes, which complicates phenotypic analyses. Here, a method to optically record action potentials specifically from ventricular-like iPSC-CMs is presented. This is achieved by lentiviral transduction with a construct in which a genetically-encoded voltage indicator is under the control of a ventricular-specific promoter element. When iPSC-CMs are transduced with this construct, the voltage sensor is expressed exclusively in ventricular-like cells, enabling subtype-specific optical membrane potential recordings using time-lapse fluorescence microscopy.

Here we present a method to optically image action potentials, specifically in ventricular-like induced pluripotent stem cell-derived cardiomyocytes. The method is based on the promoter-driven expression of a voltage-sensitive fluorescent protein.