Diese Arbeit wurde unterstützt durch das Deutsche Zentrum für Herz-Kreislauf-Forschung (DZHK; 81X3600221bis H.V., 81X2600255 bis S.C.), den China Scholarship Council (CSC201808130158 an R.X.), die Deutsche Forschungsgemeinschaft (DFG; Clinician Scientist Program in Vascular Medicine (PRIME), MA 2186/14-1 bis P. T.) und der Corona Foundation (S199/10079/2019 bis S. C.).

Enthüllung der myokardialen Architektur der Lungenvenen der Maus

<ol>
	<li>Lippi, G., Sanchis-Gomar, F., Cervellin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+epidemiology+of+atrial+fibrillation:+An+increasing+epidemic+and+public+health+challenge.">Global epidemiology of atrial fibrillation: An increasing epidemic and public health challenge.</a> International Journal of Stroke. 16 (2), 217-221 (2021).</li><li>Wijesurendra, R. S., Casadei, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+atrial+fibrillation.">Mechanisms of atrial fibrillation.</a> Heart. 105 (24), 1860-1867 (2019).</li><li>Ha&#239;ssaguerre, 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=Spontaneous+initiation+of+atrial+fibrillation+by+ectopic+beats+originating+in+the+pulmonary+veins.">Spontaneous initiation of atrial fibrillation by ectopic beats originating in the pulmonary veins.</a> The New England Journal of Medicine. 339 (10), 659-666 (1998).</li><li>Mueller-Hoecker, 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=Of+rodents+and+humans:+a+light+microscopic+and+ultrastructural+study+on+cardiomyocytes+in+pulmonary+veins.">Of rodents and humans: a light microscopic and ultrastructural study on cardiomyocytes in pulmonary veins.</a> International Journal of Medical Sciences. 5 (3), 152-158 (2008).</li><li>Kramer, A. W., Marks, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+occurrence+of+cardiac+muscle+in+the+pulmonary+veins+of+Rodenita.">The occurrence of cardiac muscle in the pulmonary veins of Rodenita.</a> Journal of Morphology. 117 (2), 135-149 (1965).</li><li>Nathan, H., Eliakim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+junction+between+the+left+atrium+and+the+pulmonary+veins.">The junction between the left atrium and the pulmonary veins.</a> Circulation. 34 (3), 412-422 (1966).</li><li>Nathan, H., Gloobe, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+atrio-venous+junctions+and+extensions+(sleeves)+over+the+pulmonary+and+caval+veins:+Anatomical+observations+in+various+mammals.">Myocardial atrio-venous junctions and extensions (sleeves) over the pulmonary and caval veins: Anatomical observations in various mammals.</a> Thorax. 25 (3), 317-324 (1970).</li><li>Bond, R. C., Choisy, S. C., Bryant, S. M., Hancox, J. C., James, 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=Ion+currents,+action+potentials,+and+noradrenergic+responses+in+rat+pulmonary+vein+and+left+atrial+cardiomyocytes.">Ion currents, action potentials, and noradrenergic responses in rat pulmonary vein and left atrial cardiomyocytes.</a> Physiological Reports. 8 (9), 14432 (2020).</li><li>Thiagalingam, A., 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=Pulmonary+vein+contraction:+Characterization+of+dynamic+changes+in+pulmonary+vein+morphology+using+multiphase+multislice+computed+tomography+scanning.">Pulmonary vein contraction: Characterization of dynamic changes in pulmonary vein morphology using multiphase multislice computed tomography scanning.</a> Heart Rhythm. 5 (12), 1645-1650 (2008).</li><li>Spach, M. S., Barr, R. C., Jewett, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spread+of+excitation+from+the+atrium+into+thoracic+veins+in+human+beings+and+dogs.">Spread of excitation from the atrium into thoracic veins in human beings and dogs.</a> The American Journal of Cardiology. 30 (8), 844-854 (1972).</li><li>Scott McNutt, N., Weinstein, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+ultrastructure+at+mammalian+intercellular+junctions.">Membrane ultrastructure at mammalian intercellular junctions.</a> Progress in Biophysics and Molecular Biology. 26, 45-101 (1973).</li><li>Barr, L., Dewey, M. M., Berger, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagation+of+action+potentials+and+the+structure+of+the+nexus+in+cardiac+muscle.">Propagation of action potentials and the structure of the nexus in cardiac muscle.</a> Journal of General Physiology. 48 (5), 797-823 (1965).</li><li>Kawamura, K., Konishi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructure+of+the+cell+junction+of+heart+muscle+with+special+reference+to+its+functional+significance+in+excitation+conduction+and+to+the+concept+of+&amp;#34;disease+of+intercalated+disc&amp;#34.">Ultrastructure of the cell junction of heart muscle with special reference to its functional significance in excitation conduction and to the concept of &amp;#34;disease of intercalated disc&amp;#34.</a> Japanese Circulation Journal. 31 (11), 1533-1543 (1967).</li><li>Van Kempen, M. J., Fromaget, C., Gros, D., Moorman, A. F., Lamers, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+distribution+of+connexin43,+the+major+cardiac+gap+junction+protein,+in+the+developing+and+adult+rat+heart.">Spatial distribution of connexin43, the major cardiac gap junction protein, in the developing and adult rat heart.</a> Circulation Research. 68 (6), 1638-1651 (1991).</li><li>Verheule, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+structure+and+connexin+expression+of+canine+pulmonary+veins.">Tissue structure and connexin expression of canine pulmonary veins.</a> Cardiovascular Research. 55 (4), 727-738 (2002).</li><li>Xiao, 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=Expression+of+connexin+43,+ion+channels+and+Ca2+-handling+proteins+in+rat+pulmonary+vein+cardiomyocytes.">Expression of connexin 43, ion channels and Ca2+-handling proteins in rat pulmonary vein cardiomyocytes.</a> Experimental and Therapeutic Medicine. 12 (5), 3233-3241 (2016).</li><li>Xia, 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=Whole-mount+immunofluorescence+staining,+confocal+imaging+and+3D+reconstruction+of+the+sinoatrial+and+atrioventricular+node+in+the+mouse.">Whole-mount immunofluorescence staining, confocal imaging and 3D reconstruction of the sinoatrial and atrioventricular node in the mouse.</a> Journal of Visualized Experiments. (166), (2020).</li><li>Tomsits, P., 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=Medetomidine/midazolam/fentanyl+narcosis+alters+cardiac+autonomic+tone+leading+to+conduction+disorders+and+arrhythmias+in+mice.">Medetomidine/midazolam/fentanyl narcosis alters cardiac autonomic tone leading to conduction disorders and arrhythmias in mice.</a> Lab Animal. 52 (4), 85-92 (2023).</li><li>Xia, 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=Isolation+and+culture+of+resident+cardiac+macrophages+from+the+murine+sinoatrial+and+atrioventricular+node.">Isolation and culture of resident cardiac macrophages from the murine sinoatrial and atrioventricular node.</a> Journal of Visualized Experiments. (171), (2021).</li><li>Bredeloux, P., Pasqualin, C., Bordy, R., Maupoil, V., Findlay, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+activity+arising+in+cardiac+muscle+sleeves+of+the+pulmonary+vein.">Automatic activity arising in cardiac muscle sleeves of the pulmonary vein.</a> Biomolecules. 12 (1), 23 (2021).</li><li>Chen, P. S., 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+mechanisms+of+atrial+fibrillation.">The mechanisms of atrial fibrillation.</a> Journal of Cardiovascular Electrophysiology. 17, S2-S7 (2006).</li><li>Thibault, S., Ton, A. -. T., Huynh, F., Fiset, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexin+lateralization+contributes+to+male+susceptibility+to+atrial+fibrillation.">Connexin lateralization contributes to male susceptibility to atrial fibrillation.</a> International Journal of Molecular Sciences. 23 (18), 10696 (2022).</li></ol>

Die Autoren haben keine Interessenkonflikte zu deklarieren.

Mit diesem Protokoll teilen wir eine Methode zur Unterscheidung und Isolierung der PVs des Mausherzens und führen eine Immunfluoreszenzfärbung durch. Nach der Organentnahme wurden Herz und Lunge in sterilisierter Saccharoselösung dehydriert, gefolgt von der Trennung der Ventrikel vom Vorhof und den Lungenlappen unter mikroskopischer Kontrolle. Danach wurde die Herzbasis vorbereitet, um die PVs zu visualisieren und sie anschließend am Hilum aus der Lunge zu schneiden. Die anschließende Immunfluoreszenzfärbung wurde ...

Vorhofflimmern ist die häufigste anhaltende Herzrhythmusstörung1. Die Prävalenz von Vorhofflimmern nimmt mit einer erwarteten Zahl von ~17,9 Millionen Patienten in Europa im Jahr 2060 noch weiter zu1. Vorhofflimmern ist klinisch von großer Bedeutung, da es ein wesentlicher Risikofaktor für die Entwicklung von Myokardinfarkt, Herzinsuffizienz oder Schlaganfall ist, was zu einer enormen individuellen, sozialen und sozioökonomischen Belastung führt1. Obwohl Vorhofflimmern seit Jahrzehnten bekannt ist, ist die Pathophysiologie von Vorhofflimmern immer noch nicht vollständig verstanden2.
Bereits in den späten 1990er Jahren zeigten Studien den großen Einfluss von Pulmonalvenen (PVs) auf die Initiierung und Aufrechterhaltung von Vorhofflimmern, da sie die Hauptquelle für Vorhofflimmern auslösende ektopische Schläge sind3. Es konnte gezeigt werden, dass sich PVs strukturell von anderen Blutgefäßen unterscheiden. Während typische Blutgefäße glatte Muskelzellen enthalten, enthält die Tunica media von PVs auch Kardiomyozyten4. Bei Nagetieren ist diese Herzmuskulatur ubiquitär in den gesamten PVs, einschließlich der intra- und extrapulmonalen Anteile, sowie der Öffnungsregion5 vorhanden. Beim Menschen enthalten PVs auch Kardiomyozyten, die innerhalb von Ausläufern des linken Vorhofmyokards (LA) beobachtet werden können - sogenannte Myokardhülsen (MS)6,7.
MS weisen morphologische Ähnlichkeiten mit dem Vorhofmyokard8 auf. Die Form und Größe von Vorhof- und PV-Kardiomyozyten unterscheidet sich nicht signifikant untereinander und zeigt vergleichbare elektrophysiologische Eigenschaften8. Elektrophysiologische Aufzeichnungen innerhalb des PV haben die elektrische Aktivität der MS nachgewiesen, und angiographische Bildgebung hat Kontraktionen gezeigt, die mit dem Herzschlag synchronisiertsind 9,10.
Gap Junctions sind porenbildende Proteinkomplexe, die aus sechs Connexin-Untereinheiten bestehen und den Durchgang von Ionen und kleinen Molekülen ermöglichen11. In den Zell-zu-Zell-Appositionen existieren Gap Junctions, die benachbarte Kardiomyozyten miteinander verbinden und eine interzelluläre elektrische Kopplung zwischen Kardiomyozytenermöglichen 12,13. Im Herzen werden mehrere Connexin-Isoformen exprimiert, wobei Connexin 43 (Cx43) die häufigste Isoform ist, die in allen Regionen des Herzens exprimiertwird 14. Frühere Studien liefern Hinweise auf die Expression von Cx43 in Kardiomyozyten der PVs15,16.
Die Untersuchung von MS in intakten PVs ist aufgrund ihrer empfindlichen Struktur, insbesondere in Kleintiermodellen, nach wie vor eine Herausforderung. In dieser Arbeit zeigen wir, wie PVs zusammen mit LA und Lungenlappen in Mäusen mittels mikroskopiegesteuerter Mikrodissektion identifiziert und isoliert werden können. Darüber hinaus demonstrieren wir die Immunfluoreszenzfärbung (IF) von PVs, um Kardiomyozyten und ihre Verbindungen innerhalb der PVs sichtbar zu machen.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adhesion slides</td>
			<td>Epredia</td>
			<td>10149870</td>
			<td></td>
		</tr>
		<tr>
			<td>AF568-secondary antibody</td>
			<td>Invitrogen</td>
			<td>A11036</td>
			<td>Host: Goat, Reactivity: Rabbit</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Biozym LE</td>
			<td>840104</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488-secondary antibody</td>
			<td>Cell Signaling Technology</td>
			<td>4408S</td>
			<td>Host: Goat, Reactivity: Mouse</td>
		</tr>
		<tr>
			<td>Anti-Connexin 43 /GJA1 antibody</td>
			<td>Abcam</td>
			<td>ab11370</td>
			<td>Polyclonal Antibody, Clone: GJA1, Host: Rabbit&#160;</td>
		</tr>
		<tr>
			<td>Anti-cTNT antibody</td>
			<td>Invitrogen</td>
			<td>MA5-12960</td>
			<td>Monoclonal Antibody, Clone: 13-11, Host: Mouse</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Brush</td>
			<td>Lukas&#160;</td>
			<td>5486</td>
			<td>size 6</td>
		</tr>
		<tr>
			<td>Cover slips</td>
			<td>Epredia</td>
			<td></td>
			<td>24 mm x 50 mm</td>
		</tr>
		<tr>
			<td>Cryotome Cryo Star NX70</td>
			<td>Epredia&#160;</td>
			<td></td>
			<td>Settings: Specimen temperature: -18 &#176;C, Blade Temperature: -25 &#176;C</td>
		</tr>
		<tr>
			<td>DFC365FX camera</td>
			<td>Leica&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DM6 B fluorescence microscope</td>
			<td>Leica&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dry ice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dubecco&#39;s phosphate-buffered saline (DPBS) 1x conc.</td>
			<td>Gibco</td>
			<td>14040133</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Dumont #5FS Forceps</td>
			<td>F.S.T.</td>
			<td>91150-20</td>
			<td>2 pieces needed</td>
		</tr>
		<tr>
			<td>Fine Scissors</td>
			<td>F.S.T.</td>
			<td>14090-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence mounting medium</td>
			<td>DAKO</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr>
			<td>Graefe Forceps</td>
			<td>F.S.T.</td>
			<td>11052-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td>Cell nuclei counterstaining</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>FIJI</td>
			<td></td>
			<td>analysis and processing software</td>
		</tr>
		<tr>
			<td>LAS X</td>
			<td>Leica&#160;</td>
			<td></td>
			<td>Imaging software for Leica DM6 B</td>
		</tr>
		<tr>
			<td>Microtome blades S35</td>
			<td>Feather</td>
			<td>207500000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microwave</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Sigma-Aldrich</td>
			<td>S26-M</td>
			<td></td>
		</tr>
		<tr>
			<td>O.C.T. compound</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde 16%</td>
			<td>Pierce</td>
			<td>28908</td>
			<td>methanol-free</td>
		</tr>
		<tr>
			<td>Pasteur pipettes</td>
			<td>VWR</td>
			<td>612-1681</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>TPP</td>
			<td>93100</td>
			<td>100 mm diameter</td>
		</tr>
		<tr>
			<td>Rocker 3D digital</td>
			<td>IKA Sch&#252;ttler</td>
			<td>00040010000</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide staining jars</td>
			<td>EasyDip</td>
			<td>M900-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Specimen Molds</td>
			<td>Tissue-Tek Cryomold</td>
			<td>4557</td>
			<td>25 mm x 20 mm x 5 mm</td>
		</tr>
		<tr>
			<td>StainTray M920 staining system</td>
			<td>StainTray</td>
			<td>631-1923</td>
			<td>Staining system for 20 slides</td>
		</tr>
		<tr>
			<td>Sterican Needle</td>
			<td>Braun</td>
			<td>4657705</td>
			<td>G 27 - used for injection (step 2) and pinning (step 3 and 4) in the protocol</td>
		</tr>
		<tr>
			<td>Student Vannas Spring Scissors</td>
			<td>F.S.T.</td>
			<td>91500-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Super PAP Pen Liquid Blocker</td>
			<td>Super PAP Pen</td>
			<td>N71310-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td>Braun</td>
			<td>4606108V</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Roche</td>
			<td>TRIS-RO</td>
			<td>component for 1x Tris-Buffered Saline (TBS)</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P2287</td>
			<td></td>
		</tr>
	</tbody>
</table>

microdissection and immunofluorescence staining of myocardial sleeves in murine pulmonary veins

Pulmonalvenen (PVs) sind die Hauptursache für ektopische Schläge bei Vorhofrhythmusstörungen und spielen eine entscheidende Rolle bei der Entstehung und dem Fortschreiten von Vorhofflimmern (VHF). PVs enthalten Myokardhülsen (MS), die aus Kardiomyozyten bestehen. MS ist an der Initiierung und Aufrechterhaltung von Vorhofflimmern beteiligt, da sie Ähnlichkeiten mit dem kardialen Arbeitsmyokard bewahrt, einschließlich der Fähigkeit, ektopische elektrische Impulse zu erzeugen. Nagetiere sind weit verbreitet und können hervorragende Tiermodelle für die Untersuchung des Lungenvenenmyokards darstellen, da Kardiomyozyten überall in der Gefäßwand vorhanden sind. Die präzise Mikrodissektion und Präparation von murinen PVs ist jedoch aufgrund der geringen Organgröße und der komplizierten Anatomie eine Herausforderung.
Wir demonstrieren ein mikroskopiegesteuertes Mikrodissektionsprotokoll zur Isolierung des murinen linken Vorhofs (LA) zusammen mit den PVs. Eine Immunfluoreszenzfärbung mit kardialen Troponin-T (cTNT) und Connexin 43 (Cx43) Antikörpern wird durchgeführt, um die LA und PVs in voller Länge sichtbar zu machen. Die Bildgebung mit 10-facher und 40-facher Vergrößerung bietet einen umfassenden Blick auf die PV-Struktur sowie detaillierte Einblicke in die myokardiale Architektur, wobei insbesondere das Vorhandensein von connexin 43 im MS hervorgehoben wird.

Atrial fibrillation (AF) is the most common sustained arrhythmia1. The prevalence of AF is increasing even further with an expected number of ~17.9 million patients in Europe in 20601. AF is clinically highly important since it is an essential risk factor for the development of myocardial infarction, heart failure, or stroke, resulting in an enormous individual, social, and socioeconomic burden1. Even though AF has been known for decades, the pathophysiology of AF is still not fully understood2.
Already in the late 1990s, studies demonstrated the great impact of pulmonary veins (PVs) in initiating and maintaining AF, as they are the main source of AF-triggering ectopic beats3. It has been demonstrated that PVs structurally differ from other blood vessels. While typical blood vessels contain smooth muscle cells, the tunica media of PVs also contains cardiomyocytes4. In rodents, this cardiac musculature is ubiquitously present throughout the whole PVs, including intra- and extrapulmonary parts, as well as the orifice region5. In humans, PVs also contain cardiomyocytes, which can be observed within extensions of the left atrial (LA) myocardium-so-called myocardial sleeves (MS)6,7.
MS have morphological similarities to the atrial myocardium8. The shape and size of atrial and PV cardiomyocytes do not vary significantly between each other and show comparable electrophysiological properties8. Electrophysiologic recordings within the PV have proven the electrical activity of MS, and angiographic imaging has revealed contractions synchronized with the heartbeat9,10.
Gap junctions are pore-forming protein complexes composed of six connexin subunits, which allow the passage of ions and small molecules11. Gap junctions exist in the cell-to-cell appositions, interconnect neighboring cardiomyocytes, and enable an intercellular electrical coupling between cardiomyocytes12,13. Several connexin isoforms are expressed in the heart with connexin 43 (Cx43) being the most common isoform expressed in all regions of the heart14. Previous studies provide evidence for the expression of Cx43 in cardiomyocytes of the PVs15,16.
It remains challenging to investigate MS within intact PVs due to their delicate structure, especially in small animal models. Here, we demonstrate how to identify and isolate PVs together with LA and lung lobes in mice using microscopy-guided microdissection. Additionally, we demonstrate immunofluorescence (IF) staining of PVs to visualize cardiomyocytes and their interconnections within the PVs.

Autor im Rampenlicht: Entwicklung eines translationalen Modells für die Speziesforschung zu Vorhofflimmern 

Mikrodissektion und Immunfluoreszenzfärbung von Myokardhülsen in murinen Pulmonalvenen

Atrial fibrillation is the most common arrhythmia, but the complex pathophysiology is still not fully understood. With our research, we contribute to a better understanding of the mechanisms that lead to the initiation and maintenance of atrial fibrillation. Translating medical research from bench to bedside is a major challenge.To address this, we established a translational pipeline from cellular models over mouse models to large animal models and pigs, allowing us to identify and validate novel therapeutic targets. Mice are often used for studying arrhythmia. However, it is challenging to study the pulmonary veins in mice.The pulmonary veins are the main triggers for atrial fibrillation in patients. Our protocol provides an effective guideline for identifying and micro-dissecting pulmonary veins to conduct further investigations.

Wir führten die Mikrodissektion, Färbung und Bildgebung der PVs in 10 12-16 Wochen alten Mäusen durch. Dem Protokoll folgend, haben wir in allen experimentellen Mäusen erfolgreich PVs zusammen mit dem LA mikrodiseziert und Schnitte mit einer umfassenden Ansicht der PVs in acht Mäusen erhalten. Übersichtsbilder wurden mit 10-facher Vergrößerung aufgenommen, um die PV-Öffnung (PVO) am LA-PV-Übergang, die extrapulmonalen PVs (PVex) (PVs zwischen dem Lungenhilum und dem LA-PV-Übergang) und die intrapulm...

Watch this Scientific Journal Video about Developing a Translational Model for Atrial Fibrillation Research Across Species at JoVE.com

Dieses Protokoll demonstriert die mikroskopiegesteuerte Isolierung und Immunfluoreszenzfärbung von murinen Pulmonalvenen. Wir bereiten Gewebeproben vor, die den linken Vorhof, die Lungenvenen und die entsprechende Lunge enthalten, und färben sie für kardiales Troponin T und Connexin 43.

Pulmonary veins (PVs) are the major source of ectopic beats in atrial arrhythmias and play a crucial role in the development and progression of atrial ...

Vorhofflimmern ist die häufigste Herzrhythmusstörung, aber die komplexe Pathophysiologie ist noch nicht vollständig verstanden. Mit unserer Forschung tragen wir zu einem besseren Verständnis der Mechanismen bei, die zur Entstehung und Aufrechterhaltung von Vorhofflimmern führen. Die Übertragung medizinischer Forschung vom Labor auf das Krankenbett ist eine große Herausforderung.Um dieses Problem anzugehen, haben wir eine translationale Pipeline von zellulären Modellen über Mausmodelle bis hin zu Großtiermodellen und Schweinen aufgebaut, die es uns ermöglicht, neue therapeutische Ziele zu identifizieren und zu validieren. Mäuse werden häufig zur Untersuchung von Herzrhythmusstörungen verwendet. Es ist jedoch schwierig, die Lungenvenen bei Mäusen zu untersuchen.Die Lungenvenen sind die Hauptauslöser für Vorhofflimmern bei Patienten. Unser Protokoll bietet eine effektive Richtlinie für die Identifizierung und Mikrodissektion von Lungenvenen zur Durchführung weiterer Untersuchungen.

microdissection-immunofluorescence-staining-myocardial-sleeves-murine

Research

JoVE Journal

Medicine

Microdissection and Immunofluorescence Staining of Myocardial Sleeves in Murine Pulmonary Veins

513 Aufrufe.  Ludwig-Maximilians-University Munich (LMU). Vorhofflimmern ist die häufigste Herzrhythmusstörung, aber die komplexe Pathophysiologie ist noch nicht vollständig verstanden. Mit unserer Forschung tragen wir zu einem besseren Verständnis der Mechanismen bei, die zur Entstehung und Aufrechterhaltung von Vorhofflimmern führen. Die Übertragung medizinischer Forschung vom Labor auf das Krankenbett ist eine große Herausforderung.Um dieses Problem anzugehen, haben wir eine translationale Pipeline von zellulären Modellen über Mau...

Serie: Autor im Rampenlicht: Entwicklung eines translationalen Modells für die Speziesforschung zu Vorhofflimmern 

Pulmonary veins (PVs) are the major source of ectopic beats in atrial arrhythmias and play a crucial role in the development and progression of atrial fibrillation (AF). PVs contain myocardial sleeves (MS) composed of cardiomyocytes. MS are implicated in the initiation and maintenance of AF, as they preserve similarities to the cardiac working myocardium, including the ability to generate ectopic electrical impulses. Rodents are widely used and may represent excellent animal models to study the pulmonary vein myocardium since cardiomyocytes are widely present all over the vessel wall. However, precise microdissection and preparation of murine PVs is challenging due to the small organ size and intricate anatomy.
We demonstrate a microscopy-guided microdissection protocol for isolating the murine left atrium (LA) together with the PVs. Immunofluorescence staining using cardiac Troponin-T (cTNT) and connexin 43 (Cx43) antibodies is performed to visualize the LA and PVs in full length. Imaging at 10x and 40x magnification provides a comprehensive view of the PV structure as well as detailed insights into the myocardial architecture, particularly highlighting the presence of connexin 43 within the MS.

This protocol demonstrates microscopy-guided isolation and immunofluorescence staining of murine pulmonary veins. We prepare tissue samples containing the left atrium, pulmonary veins, and the corresponding lungs and stain them for cardiac Troponin T and Connexin 43.

Forschung

Medizin

Microdissection Protocol for Isolating the Murine Left Atrium (LA) with the Pulmonary Veins

To begin, place the dehydrated heart and lungs isolated from a mouse into a dissection dish containing 40 to 50 milliliters of PBS. Position it under a bright-field microscope with 10x magnification and set up the lighting. Then place the heart's dorsal surface on the dissection dish.Using surgical scissors, carefully separate the ventricles from the atria after identifying the transition between the ventricular and atrial wall. Place the separated atria on the dissection dish with the heart base facing up. Orient the atria with the lung lobes positioned posteriorly, the left atrium and its appendage situated on the right and the right atrium and its appendage situated on the left.Pin the remaining left ventricular tissue to the dissection dish to secure the preparation. Spread the lung lobes gently without exerting excessive force and pin them to the dissection dish. Next, find the aorta with the aortic arch in the center of the heart base.Remove connective and fat tissue around the heart base while preserving all identified landmarks. Also, remove the aortic arch to clear the view of the heart base. Detect the pulmonary trunk directly to the right of the aorta and trace its path toward the back to identify the pulmonary arteries.To confirm their position, track their route to the left lobe and right superior or middle lobes to pinpoint the lung hilum. Determine the location of the left and right main bronchi behind the aorta. Confirm their position by tracing their path towards the lung hilum.Then identify the superior vena cava to the left of the aorta and trace its path into the right atrium near the right atrial appendage. Using blunt dissection with fine forceps, carefully detach the pulmonary arteries from the atrium's top surface, then sever them in the lung hilum and move them aside to unveil the left atrial free wall and the pulmonary veins in full view. Remove the main bronchi from the lung hilum and discard the bronchial tissue.Examine the lung hilum to identify the ostium of the pulmonary artery and the main bronchus. To separate the atria and ventricles, make a cut from the front part of the remaining right ventricle and move upward through the tricuspid valve to the front side of the vena cava superior, opening the right atrium from the frontal side. Then cut the posterior right atrial free wall adjacent to the interatrial septum to create a division between the right and left atrium.Cut the posterior right ventricular wall next to the interventricular septum to completely disconnect the left and right ventricles. Remove some of the basal lung tissue to trim the size of the lung lobes, leaving three to four millimeters of the lung tissue.

Mikrodissektionsprotokoll zur Isolierung des linken Vorhofs (LA) der Maus mit den Pulmonalvenen

Tissue Embedding and Immunofluorescence Staining of Myocardial Sleeves in Murine Pulmonary Veins

To begin, isolate the left atrium and pulmonary veins or PVs from the mouse's heart. Place the isolated tissue into a cryomold with the heart base and lung apex facing up. Untwist and untangle the PVs in case they are convoluted.Then, fill the cryomold with the OCT compound. Using fine forceps, apply gentle compression on the PVs without moving them to remove residual air. Freeze the tissue in the OCT compound on dry ice.For immunostaining, thaw the frozen mouse PV slides on a staining system for 20 minutes. Then administer several drops of a 4%paraformaldehyde-fixing solution onto the sections to secure the tissue to the slide. After incubation, move the slides to a vertical rack and submerge them in a container filled with PBS.Set the container on a slow shaker for five minutes to rinse the slides. Using a liquid blocker pen containing xylol, encircle each section on every slide. And reorganize the slides on the staining system.Using a Pasteur pipette, apply one or two drops of permeabilization solution onto each sample. Then, clean the Triton X-100 solution by washing the slides for five minutes in PBS. After rearranging the slides on the staining system, add one or two drops of the blocking buffer to each section, ensuring complete coverage.Next, prepare one milliliter of the primary antibody mix consisting of the primary antibodies and blocking buffer. Discard the leftover blocking buffer from the slide. Administer two or three drops of the primary antibody mixture to each section.After incubation, place the slides in a vertical rack and rinse the slides for five minutes in wash buffer while maintaining gentle agitation. Then, prepare one milliliter of the secondary antibody mix consisting of the secondary antibodies and washing buffer. Reposition the slides on the staining system and use a pipette to dispense two or three drops of the secondary antibody blend onto each section.Subsequently, clean the slides three times for five minutes each in wash buffer while shaking. Next, add two or three drops of the Hoechst 33342 solution to each section arranged on the staining system. Place the slides in a vertical rack and rinse them for five minutes in wash buffer with gentle agitation.Add one or two drops of fluorescent mounting medium to each slide and seal the slides with cover slips. The pulmonary vein orifice region at the left atrium and pulmonary vein junction, extrapulmonary veins, and interpulmonary veins were observed under 10X magnification. Specific cTnT signals with a typical muscular striation in the pulmonary vein orifice, extrapulmonary veins, and intrapulmonary veins, were observed at 40X magnification.Cx43 was found in all three pulmonary regions and was primarily projected between neighboring cardiomyocytes. Cx43-related signals were observed on the polar side and the lateral side of the cardiomyocytes in the myocardial sleeves.