이 연구는 독일 심혈관 연구 센터(DZHK; 81X3600221to H.V., 81X2600255 to S.C.), 중국 장학위원회(CSC201808130158 to R.X.), 독일 연구 재단(DFG; 혈관 의학 임상 과학자 프로그램(PRIME), MA 2186/14-1 to P. T.), Corona Foundation(S199/10079/2019 to SC).

Revealing Murine Pulmonary Veins Myocardial Architecture

<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>

저자는 선언할 이해 상충이 없습니다.

이 프로토콜을 통해 쥐 심장의 PV를 구별 및 분리하고 면역형광 염색을 수행하는 방법을 공유합니다. 장기 적출 후, 심장과 폐를 멸균된 자당 용액에서 탈수한 후, 현미경 유도 하에 심방과 폐엽에서 심실을 분리하였다. 그 후, 심장 베이스는 PV를 시각화하기 위해 준비되었으며, 그 후 hilum에서 폐에서 PV를 절단했습니다. 후속 면역형광 염색은 조직을 O.C.T. 화합물에 매립하고, 빙정체로 절단하고, cT...

심방세동(AF)은 가장 흔한 지속 부정맥이다1. 심방세동 유병률은 더욱 증가하여 2060년에는 유럽에서 ~1,790만 명의 환자가 발생할 것으로 예상됩니다1. 심방세동은 심근경색, 심부전 또는 뇌졸중의 발병에 필수적인 위험 요소이기 때문에 임상적으로 매우 중요하며, 이는 엄청난 개인적, 사회적, 사회경제적 부담을 초래한다1. 심방세동은 수십 년 동안 알려져 왔지만, 심방세동의 병태생리학은 아직 완전히 이해되지 않았다2.
이미 1990년대 후반에 여러 연구에서 폐정맥(PV)이 심방세동을 유발하는 이소성 박동(ectopic beats)의 주요 원인이기 때문에 심방세동을 시작하고 유지하는 데 큰 영향을 미친다는 사실이 입증되었습니다3. PV는 구조적으로 다른 혈관과 다르다는 것이 입증되었습니다. 일반적인 혈관에는 평활근 세포가 포함되어 있지만, PV의 튜니카 배지에는 심근세포도 포함되어 있다4. 설치류에서, 이 심장 근육계는 폐내 및 폐외 부분뿐만 아니라 오리피스 영역(5)을 포함한 전체 PV에 걸쳐 어디에나 존재한다. 인간의 경우 PV에는 좌심방(LA) 심근 슬리브(MS)6,7라고 하는 심근의 확장 내에서 관찰할 수 있는 심근세포도 포함되어 있습니다.
다발성경화증은 심방심근(atrial myocardium8)과 형태학적 유사성을 가지고 있다. 심방과 PV 심근세포의 모양과 크기는 서로 크게 다르지 않으며 유사한 전기생리학적 특성을 나타낸다8. PV 내의 전기 생리학적 기록은 다발성경화증의 전기적 활성을 입증했으며, 혈관 조영술은 심장 박동 9,10과 동기화된 수축을 밝혀냈다.
간극연접(gap junction)은 6개의 커넥신 소단위체(connexin subunit)로 구성된 기공 형성 단백질 복합체로, 이온과 소분자(11)의 통과를 허용한다. 간극연접(gap junction)은 세포-세포 접합부(cell-to-cell appositions)에 존재하고, 이웃한 심근세포를 상호 연결하며, 심근세포(cardiomyocyte) 사이의 세포간 전기적 결합을 가능하게 한다(12,13). 여러 개의 커넥신 동형이 심장에서 발현되며, 커넥신 43(Cx43)은 심장의 모든 영역에서 발현되는 가장 흔한 동형이다14. 이전 연구는 PVs15,16의 심근세포에서 Cx43의 발현에 대한 증거를 제공합니다.
온전한 PV의 섬세한 구조로 인해, 특히 작은 동물 모델에서 MS를 조사하는 것은 여전히 어렵습니다. 여기에서는 현미경 유도 미세해부를 사용하여 마우스에서 LA 및 폐엽과 함께 PV를 식별하고 분리하는 방법을 보여줍니다. 또한 PV의 면역형광(IF) 염색을 시연하여 심근세포와 PV 내 상호 연결을 시각화합니다.

<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

폐정맥(PV)은 심방 부정맥에서 이소성 박동의 주요 원인이며 심방세동(AF)의 발생과 진행에 중요한 역할을 합니다. PV에는 심근세포로 구성된 심근 슬리브(MS)가 포함되어 있습니다. 다발성경화증은 이소성 전기 충격을 생성하는 능력을 포함하여 심장 작동 심근과의 유사성을 보존하기 때문에 심방세동의 시작 및 유지와 관련이 있습니다. 설치류는 널리 사용되며 심근세포가 혈관 벽 전체에 널리 존재하기 때문에 폐 정맥 심근을 연구하는 데 우수한 동물 모델을 나타낼 수 있습니다. 그러나 쥐 PV의 정확한 미세해부 및 준비는 작은 장기 크기와 복잡한 해부학적 구조로 인해 어렵습니다.
PV와 함께 쥐 좌심방(LA)을 분리하기 위한 현미경 유도 현세해부 프로토콜을 시연합니다. 심장 Troponin-T(cTNT) 및 connexin 43(Cx43) 항체를 사용한 면역형광 염색을 수행하여 LA 및 PV를 전체 길이로 시각화합니다. 10x 및 40x 배율의 이미징은 PV 구조에 대한 포괄적인 보기와 심근 구조에 대한 자세한 통찰력을 제공하며, 특히 MS 내에서 connexin 43의 존재를 강조합니다.

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.

저자 스포트라이트: Developing a Translational Model for Atrial Fibrillation Research Across Species(종 간 심방세동 연구를 위한 중개 모델 개발) 

쥐 폐 정맥에서 심근 슬리브의 미세 해부 및 면역형광 염색

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.

우리는 12-16주 된 쥐 10마리에서 PV의 미세 해부, 염색 및 이미징을 수행했습니다. 프로토콜에 따라 모든 실험용 마우스에서 LA와 함께 PV를 성공적으로 미세 해부하고 8개 마우스에서 PV를 포괄적으로 볼 수 있는 섹션을 얻었습니다. 개요 이미지는 LA-PV 접합부의 PV 오리피스(PVO) 영역, 폐 외 PV(PVex)(폐 힐럼과 LA-PV 접합부 사이의 PV) 및 폐 내 PV(PVin)(폐 조직으로 둘러싸인 PV)를 식별하기 ?...

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

이 프로토콜은 쥐 폐 정맥의 현미경 유도 분리 및 면역형광 염색을 보여줍니다. 좌심방, 폐정맥 및 해당 폐가 포함된 조직 샘플을 준비하고 심장 Troponin T 및 Connexin 43에 대해 염색합니다.

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 ...

심방세동은 가장 흔한 부정맥이지만 복잡한 병태생리학은 아직 완전히 이해되지 않았습니다. 우리의 연구를 통해 우리는 심방세동의 시작과 유지로 이어지는 메커니즘을 더 잘 이해하는 데 기여합니다. 의학 연구를 벤치에서 병상으로 옮기는 것은 중요한 과제입니다.이 문제를 해결하기 위해 우리는 마우스 모델에 대한 세포 모델에서 대형 동물 모델 및 돼지에 이르는 중개 파이프라인을 구축하여 새로운 치료 표적을 식별하고 검증할 수 있었습니다. 생쥐는 부정맥을 연구하는 데 자주 사용됩니다. 그러나 쥐의 폐 정맥을 연구하는 것은 어렵습니다.폐정맥은 환자의 심방세동을 유발하는 주요 원인입니다. 당사의 프로토콜은 추가 조사를 수행하기 위해 폐 정맥을 식별하고 미세 해부하기 위한 효과적인 지침을 제공합니다.

microdissection-immunofluorescence-staining-myocardial-sleeves-murine

Research

JoVE Journal

Medicine

Microdissection and Immunofluorescence Staining of Myocardial Sleeves in Murine Pulmonary Veins

513 조회수.  Ludwig-Maximilians-University Munich (LMU). 심방세동은 가장 흔한 부정맥이지만 복잡한 병태생리학은 아직 완전히 이해되지 않았습니다. 우리의 연구를 통해 우리는 심방세동의 시작과 유지로 이어지는 메커니즘을 더 잘 이해하는 데 기여합니다. 의학 연구를 벤치에서 병상으로 옮기는 것은 중요한 과제입니다.이 문제를 해결하기 위해 우리는 마우스 모델에 대한 세포 모델에서 대형 동물 모델 및 돼지에 이르는...

비디오: 저자 스포트라이트: Developing a Translational Model for Atrial Fibrillation Research Across Species(종 간 심방세동 연구를 위한 중개 모델 개발) 

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.

연구

의학

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.

폐 정맥으로 쥐 좌심방(LA)을 분리하기 위한 미세해부 프로토콜

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.