Questo lavoro è stato sostenuto dal Centro tedesco per la ricerca cardiovascolare (DZHK; 81X3600221to H.V., 81X2600255 to S.C.), dal China Scholarship Council (CSC201808130158 to R.X.), dalla Fondazione tedesca per la ricerca (DFG; Clinician Scientist Program in Vascular Medicine (PRIME), MA 2186/14-1 a P. T.), e la Fondazione Corona (S199/10079/2019 a S. C.).

Rivelatore dell'architettura miocardica delle vene polmonari murine

<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>

Gli autori non hanno conflitti di interesse da dichiarare.

Con questo protocollo, condividiamo un metodo per distinguere e isolare i PV del cuore di topo ed eseguire la colorazione in immunofluorescenza su di essi. Dopo il prelievo di organi, il cuore e i polmoni sono stati disidratati in una soluzione di saccarosio sterilizzata, seguita dalla separazione dei ventricoli dall'atrio e dai lobi polmonari sotto guida microscopica. Successivamente, la base del cuore è stata preparata per visualizzare i PV seguiti dal taglio dai polmoni all'ilo. La successiva colorazione a immunofluo...

La fibrillazione atriale (FA) è l'aritmia sostenuta più comune1. La prevalenza della fibrillazione atriale è in ulteriore aumento, con un numero previsto di ~17,9 milioni di pazienti in Europa nel 20601. La fibrillazione atriale è clinicamente molto importante poiché è un fattore di rischio essenziale per lo sviluppo di infarto miocardico, insufficienza cardiaca o ictus, con conseguente enorme onere individuale, sociale e socioeconomico1. Anche se la fibrillazione atriale è nota da decenni, la fisiopatologia della fibrillazione atriale non è ancora del tutto compresa2.
Già alla fine degli anni '90, gli studi hanno dimostrato il grande impatto delle vene polmonari (PV) nell'avvio e nel mantenimento della fibrillazione atriale, in quanto sono la principale fonte di battiti ectopici che innescano la fibrillazione atriale3. È stato dimostrato che i PV differiscono strutturalmente dagli altri vasi sanguigni. Mentre i vasi sanguigni tipici contengono cellule muscolari lisce, la tunica media dei PV contiene anche cardiomiociti4. Nei roditori, questa muscolatura cardiaca è presente in modo ubiquitario in tutte le PV, comprese le parti intra ed extrapolmonari, nonché la regione dell'orifizio5. Nell'uomo, i PV contengono anche cardiomiociti, che possono essere osservati all'interno delle estensioni del miocardio dell'atrio sinistro (LA), le cosiddette maniche miocardiche (SM)6,7.
La SM presenta somiglianze morfologiche con il miocardio8 atriale. La forma e le dimensioni dei cardiomiociti atriali e PV non variano significativamente tra loro e mostrano proprietà elettrofisiologiche comparabili8. Le registrazioni elettrofisiologiche all'interno del PV hanno dimostrato l'attività elettrica della SM e l'imaging angiografico ha rivelato contrazioni sincronizzate con il battito cardiaco 9,10.
Le giunzioni gap sono complessi proteici che formano pori composti da sei subunità connessine, che consentono il passaggio di ioni e piccole molecole11. Le giunzioni gap esistono nelle apposizioni cellula-cellula, interconnettono i cardiomiociti vicini e consentono un accoppiamento elettrico intercellulare tra i cardiomiociti 12,13. Diverse isoforme di connessina sono espresse nel cuore, con la connessina 43 (Cx43) che è l'isoforma più comune espressa in tutte le regioni del cuore14. Studi precedenti hanno fornito prove dell'espressione di Cx43 nei cardiomiociti dei PV15,16.
Rimane difficile studiare la SM all'interno di PV intatti a causa della loro struttura delicata, specialmente nei modelli animali di piccola taglia. Qui, dimostriamo come identificare e isolare i PV insieme a LA e ai lobi polmonari nei topi utilizzando la microdissezione guidata dalla microscopia. Inoltre, dimostriamo la colorazione in immunofluorescenza (IF) dei PV per visualizzare i cardiomiociti e le loro interconnessioni all'interno dei 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

Le vene polmonari (PV) sono la principale fonte di battiti ectopici nelle aritmie atriali e svolgono un ruolo cruciale nello sviluppo e nella progressione della fibrillazione atriale (FA). I PV contengono manicotti miocardici (SM) composti da cardiomiociti. Le sclerosi multipla sono implicate nell'inizio e nel mantenimento della fibrillazione atriale, in quanto conservano somiglianze con il miocardio cardiaco, inclusa la capacità di generare impulsi elettrici ectopici. I roditori sono ampiamente utilizzati e possono rappresentare ottimi modelli animali per studiare il miocardio della vena polmonare poiché i cardiomiociti sono ampiamente presenti in tutta la parete del vaso. Tuttavia, la microdissezione precisa e la preparazione di PV murini sono impegnative a causa delle piccole dimensioni dell'organo e dell'anatomia intricata.
Dimostriamo un protocollo di microdissezione guidato dalla microscopia per isolare l'atrio sinistro murino (LA) insieme ai PV. Viene eseguita una colorazione a immunofluorescenza utilizzando anticorpi cardiaci Troponina-T (cTNT) e connessina 43 (Cx43) per visualizzare LA e PV in tutta la sua lunghezza. L'imaging con ingrandimenti 10x e 40x fornisce una visione completa della struttura del PV e approfondimenti dettagliati sull'architettura miocardica, evidenziando in particolare la presenza della connessina 43 all'interno della SM.

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.

Autore in primo piano: Sviluppo di un modello traslazionale per la ricerca sulla fibrillazione atriale in tutte le specie 

Microdissezione e colorazione in immunofluorescenza di manicotti miocardici nelle vene polmonari murine

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.

Abbiamo eseguito la microdissezione, la colorazione e l'imaging dei PV in 10 topi di 12-16 settimane. Seguendo il protocollo, abbiamo microsezionato con successo i PV insieme al LA in tutti i topi sperimentali e abbiamo ottenuto sezioni con una visione completa dei PV in otto topi. Le immagini panoramiche sono state scattate con un ingrandimento di 10x per identificare la regione dell'orifizio PV (PVO) alla giunzione LA-PV, i PV extrapolmonari (PVex) (PV tra l'ilo polmonare e la giunzione LA-PV) e i PV intrapo...

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

Questo protocollo dimostra l'isolamento guidato dalla microscopia e la colorazione in immunofluorescenza delle vene polmonari murine. Prepariamo campioni di tessuto contenenti l'atrio sinistro, le vene polmonari e i polmoni corrispondenti e li coloriamo per la troponina T cardiaca e la connessina 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 fibrillazione atriale è l'aritmia più comune, ma la complessa fisiopatologia non è ancora del tutto compresa. Con la nostra ricerca, contribuiamo a una migliore comprensione dei meccanismi che portano all'inizio e al mantenimento della fibrillazione atriale. Tradurre la ricerca medica dal laboratorio al letto del paziente è una sfida importante.Per affrontare questo problema, abbiamo stabilito una pipeline traslazionale da modelli cellulari su modelli murini a modelli animali di grandi dimensioni e suini, che ci consente di identificare e convalidare nuovi bersagli terapeutici. I topi sono spesso usati per studiare l'aritmia. Tuttavia, è difficile studiare le vene polmonari nei topi.Le vene polmonari sono i principali fattori scatenanti della fibrillazione atriale nei pazienti. Il nostro protocollo fornisce una linea guida efficace per l'identificazione e la micro-dissezione delle vene polmonari per condurre ulteriori indagini.

microdissection-immunofluorescence-staining-myocardial-sleeves-murine

Research

JoVE Journal

Medicine

Microdissection and Immunofluorescence Staining of Myocardial Sleeves in Murine Pulmonary Veins

514 Views.  Ludwig-Maximilians-University Munich (LMU). La fibrillazione atriale è l'aritmia più comune, ma la complessa fisiopatologia non è ancora del tutto compresa. Con la nostra ricerca, contribuiamo a una migliore comprensione dei meccanismi che portano all'inizio e al mantenimento della fibrillazione atriale. Tradurre la ricerca medica dal laboratorio al letto del paziente è una sfida importante.Per affrontare questo problema, abbiamo stabilito una pipeline traslazionale da modelli cellulari su modelli murini a modelli animali di...

Video: Autore in primo piano: Sviluppo di un modello traslazionale per la ricerca sulla fibrillazione atriale in tutte le specie 

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.

Ricerca

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.

Protocollo di microdissezione per l'isolamento dell'atrio murino sinistro (LA) con le vene polmonari

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.