Name: Microdissection Protocol for Isolating the Murine Left Atrium (LA) with the Pulmonary Veins
Uploaded: 2023-11-21T13:20:00
Description: Watch this Scientific Journal Video about Microdissection Protocol for Isolating the Murine Left Atrium LA with the Pulmonary Veins at JoVE.com

microdissection protocol for isolating the murine left atrium (la) with the pulmonary veins

Revealing Murine Pulmonary Veins Myocardial Architecture

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.

Author Spotlight: Developing a Translational Model for Atrial Fibrillation Research Across Species 

Microdissection and Immunofluorescence Staining of Myocardial Sleeves in Murine Pulmonary Veins

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.

Watch this Scientific Journal Video about Microdissection Protocol for Isolating the Murine Left Atrium LA with the Pulmonary Veins at JoVE.com

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.

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Research

JoVE Journal

Medicine

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.

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 ...

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.