Este trabajo contó con el apoyo del Centro Alemán de Investigación Cardiovascular (DZHK; 81X3600221 a H.V., 81X2600255 a S.C.), el Consejo de Becas de China (CSC201808130158 a R.X.), la Fundación Alemana de Investigación (DFG; Programa de Científicos Clínicos en Medicina Vascular (PRIME), MA 2186/14-1 a P. T.), y la Fundación Corona (S199/10079/2019 a S. C.).

Revelando la arquitectura miocárdica de las venas pulmonares murinas

<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>

Los autores no tienen conflictos de intereses que declarar.

Con este protocolo, compartimos un método para distinguir y aislar las VPs del corazón del ratón y realizar tinciones de inmunofluorescencia sobre ellas. Después de la extracción de órganos, el corazón y los pulmones se deshidrataron en una solución de sacarosa esterilizada, seguida de la separación de los ventrículos de la aurícula y los lóbulos pulmonares bajo guía microscópica. Posteriormente, se preparó la base del corazón para visualizar los VP y luego se cortaron de los pulmones en el hilio. La post...

La fibrilación auricular (FA) es la arritmia sostenida más frecuente1. La prevalencia de la FA está aumentando aún más, con un número esperado de ~17,9 millones de pacientes en Europa en 20601. La FA es clínicamente muy importante, ya que es un factor de riesgo esencial para el desarrollo de infarto de miocardio, insuficiencia cardíaca o accidente cerebrovascular, lo que resulta en una enorme carga individual, social y socioeconómica1. A pesar de que la FA se conoce desde hace décadas, la fisiopatología de la FA aún no se comprende completamente2.
Ya a finales de la década de 1990, los estudios demostraron el gran impacto de las venas pulmonares (VP) en el inicio y mantenimiento de la FA, ya que son la principal fuente de latidos ectópicos desencadenantes de la FA3. Se ha demostrado que los VP difieren estructuralmente de otros vasos sanguíneos. Mientras que los vasos sanguíneos típicos contienen células musculares lisas, la túnica media de los PV también contiene cardiomiocitos4. En los roedores, esta musculatura cardíaca está presente de forma ubicua en toda la VP, incluidas las partes intra y extrapulmonares, así como en la región del orificio5. En los seres humanos, los VP también contienen cardiomiocitos, que pueden observarse dentro de las extensiones del miocardio de la aurícula izquierda (AI), las llamadas mangas miocárdicas (EM)6,7.
Los SM tienen similitudes morfológicas con el miocardio auricular8. La forma y el tamaño de los cardiomiocitos auriculares y PV no varían significativamente entre sí y muestran propiedades electrofisiológicas comparables8. Los registros electrofisiológicos dentro de la VP han demostrado la actividad eléctrica del SM y las imágenes angiográficas han revelado contracciones sincronizadas con los latidos del corazón 9,10.
Las uniones gap son complejos proteicos formadores de poros compuestos por seis subunidades de conexina, que permiten el paso de iones y moléculas pequeñas11. Existen uniones gap en las aposiciones de célula a célula, interconectan cardiomiocitos vecinos y permiten un acoplamiento eléctrico intercelular entre cardiomiocitos 12,13. Varias isoformas de conexina se expresan en el corazón, siendo la conexina 43 (Cx43) la isoforma más común expresada en todas las regiones del corazón14. Estudios previos proporcionan evidencias de la expresión de Cx43 en cardiomiocitos de los PVs15,16.
Sigue siendo un reto investigar la EM dentro de las VP intactas debido a su delicada estructura, especialmente en modelos animales pequeños. Aquí, demostramos cómo identificar y aislar los PV junto con los lóbulos pulmonares y de LA en ratones mediante microdisección guiada por microscopía. Además, demostramos la tinción por inmunofluorescencia (IF) de los PV para visualizar los cardiomiocitos y sus interconexiones dentro de los PV.

<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

Las venas pulmonares (VP) son la principal fuente de latidos ectópicos en las arritmias auriculares y desempeñan un papel crucial en el desarrollo y la progresión de la fibrilación auricular (FA). Los VP contienen mangas miocárdicas (EM) compuestas por cardiomiocitos. Los SM están implicados en el inicio y mantenimiento de la FA, ya que conservan similitudes con el miocardio cardíaco que funciona, incluida la capacidad de generar impulsos eléctricos ectópicos. Los roedores son ampliamente utilizados y pueden representar excelentes modelos animales para estudiar el miocardio de la vena pulmonar, ya que los cardiomiocitos están ampliamente presentes en toda la pared del vaso. Sin embargo, la microdisección y preparación precisas de las VP murinas es un reto debido al pequeño tamaño de los órganos y a la intrincada anatomía.
Demostramos un protocolo de microdisección guiada por microscopía para aislar la aurícula izquierda murina (AI) junto con los VPs. Se realiza una tinción de inmunofluorescencia utilizando anticuerpos cardíacos contra la troponina-T (cTNT) y la conexina 43 (Cx43) para visualizar los AL y los VPs en toda su longitud. Las imágenes con aumentos de 10x y 40x proporcionan una visión completa de la estructura de la VP, así como información detallada sobre la arquitectura miocárdica, destacando especialmente la presencia de la conexina 43 dentro de la EM.

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 destacado: Desarrollo de un modelo traslacional para la investigación de la fibrilación auricular en todas las especies 

Microdisección y tinción con inmunofluorescencia de mangas miocárdicas en venas pulmonares murinas

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.

Realizamos la microdisección, tinción e imágenes de los PV en 10 ratones de 12 a 16 semanas de edad. Siguiendo el protocolo, microdiseccionamos con éxito los VP junto con el AL en todos los ratones experimentales y obtuvimos secciones con una visión completa de los VP en ocho ratones. Se tomaron imágenes generales con un aumento de 10x para identificar la región del orificio de la VP (PVO) en la unión LA-PV, las VP extrapulmonares (PVex) (VP entre el hilio pulmonar y la unión LA-PV) y las VP intrapulm...

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

Este protocolo demuestra el aislamiento guiado por microscopía y la tinción por inmunofluorescencia de las venas pulmonares murinas. Preparamos muestras de tejido que contienen la aurícula izquierda, las venas pulmonares y los pulmones correspondientes y las teñimos para troponina T cardíaca y conexina 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 ...

La fibrilación auricular es la arritmia más común, pero la compleja fisiopatología aún no se comprende completamente. Con nuestra investigación, contribuimos a una mejor comprensión de los mecanismos que conducen al inicio y mantenimiento de la fibrilación auricular. Trasladar la investigación médica del laboratorio a la cabecera del paciente es un gran reto.Para abordar esto, establecimos una línea de traslación desde modelos celulares sobre modelos de ratón hasta modelos de animales grandes y cerdos, lo que nos permite identificar y validar nuevas dianas terapéuticas. Los ratones se utilizan a menudo para estudiar la arritmia. Sin embargo, es difícil estudiar las venas pulmonares en ratones.Las venas pulmonares son los principales desencadenantes de la fibrilación auricular en los pacientes. Nuestro protocolo proporciona una guía eficaz para identificar y microdiseccionar las venas pulmonares para realizar más investigaciones.

microdissection-immunofluorescence-staining-myocardial-sleeves-murine

Research

JoVE Journal

Medicine

Microdissection and Immunofluorescence Staining of Myocardial Sleeves in Murine Pulmonary Veins

514 vistas.  Ludwig-Maximilians-University Munich (LMU). La fibrilación auricular es la arritmia más común, pero la compleja fisiopatología aún no se comprende completamente. Con nuestra investigación, contribuimos a una mejor comprensión de los mecanismos que conducen al inicio y mantenimiento de la fibrilación auricular. Trasladar la investigación médica del laboratorio a la cabecera del paciente es un gran reto.Para abordar esto, establecimos una línea de traslación desde modelos celulares sobre modelos de ratón hasta modelos...

Video: Autor destacado: Desarrollo de un modelo traslacional para la investigación de la fibrilación auricular en todas las especies 

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.

Investigación

Medicina

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.

Protocolo de microdisección para aislar la aurícula izquierda murina (AI) con las venas pulmonares

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.