Name: Langendorff Retrograde Heart Perfusion: An Ex Vivo Technique to Perfuse Isolated Mouse Heart
Uploaded: 2023-04-30T14:50:07
Description: Source: Wu, K. et al. Modifications of the Langendorff Method for Simultaneous Isolation of Atrial and Ventricular Myocytes from Adult Mice. J. Vis. Exp. ...

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Solution and Perfusion Apparatus Preparation
<ol>
	<li>Assemble a constant flow Langendorff apparatus (commercially available or self-made).&#160;Figure 1&#160;provides a simple schematic.</li>
	<li>Prepare the solutions according to the reagents given in&#160;Table 1&#160;and&#160;Table ...

langendorff retrograde heart perfusion: an ex vivo technique to perfuse isolated mouse heart

Source: Wu, K. et al. <a href="https://www.jove.com/video/62514" target="_blank">Modifications of the Langendorff Method for Simultaneous Isolation of Atrial and Ventricular Myocytes from Adult Mice</a>. J. Vis. Exp. (2021)
In this video, we perform Langendorff retrograde perfusion of an isolated mouse heart. During the procedure, the cardiac activity of the heart is maintained by perfusing it retrogradely with a perfusion solution via the coronary arteries using a cannula inserted into the ascending aorta. The model is used to study the cardiac pathophysiology of the heart.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/20749/20749_Figure1.jpg" /> 
Figure 1. Schematic diagram of an assembled modified Langendorff system.&#160;This system is economical and portable for the users. It has two parts of plastic tubing-one for the water (inner diameter, 8 mm) and one for the perfusate (inner diameter, 1.5 mm)-of which the distal end is connected to a PE medical three-way stopcock with a Luer lock. A glass bottle containing water an...

Langendorff Retrograde Heart Perfusion: An Ex Vivo Technique to Perfuse Isolated Mouse Heart

Source: Wu, K. et al. Modifications of the Langendorff Method for Simultaneous Isolation of Atrial and Ventricular Myocytes from Adult Mice. J. Vis. Exp. ...

To begin, place an anesthetized mouse in the supine position on a dissection board. Make an incision to access the thoracic cavity and expose the heart. Locate the aorta and its branches - the left common carotid artery and brachiocephalic artery. Transect these arteries to isolate the heart.
Immediately, immerse the excised heart into a Petri dish containing a nutrient-rich oxygenated buffer solution. Concomitantly, insert a cannula into the heart such that its tip reaches the ascending aorta. Use a suture to ligate the cannula. Connect the cannula to a syringe containing oxygenated perfusion solution.
Inject the perfusion solution into the heart. This step allows the blood to leave the coronary arteries and effuse from the pulmonary veins. Next, connect the cannulated heart to the Langendorff apparatus. Use peristaltic pump to perform perfusion at a constant flow rate.
Under these conditions, the buffer perfuses into the heart. This creates cannulation pressure that closes the aortic valves, causing the buffer to move through the coronary arteries. Eventually, the buffer perfuses the heart muscles retrogradely and later effuses through the pulmonary veins. A successfully perfused heart is now ready for further downstream applications.

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Biology

Rodent Models

Adult

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and electrophysiological phenotype in the atria. That encourages us to utilize a genetically-engineered mouse model to elucidate the mechanism of AF. However, it is difficult to evaluate the electrophysiological properties in murine atria due to their small size. This protocol describes the electrophysiological evaluation of atria using an optical mapping system with a high temporal and spatial resolution in Langendorff perfused murine hearts. The optical mapping system is assembled with dual high-speed complementary metal oxide semiconductor cameras and high magnification objective lenses, to detect the fluorescence of a voltage-sensitive dye and Ca2+ indicator. To focus on the assessment of murine atria, optical mapping is performed with an area of 2 mm &#215; 2 mm or 10 mm x 10 mm, with a 100 &#215; 100 resolution (20 &#181;m/pixel or 100 &#181;m/pixel) and sampling rate of up to 10 kHz (0.1 ms) at maximum. A 1-French size quadripolar electrode pacing catheter is placed into the right atrium through the superior vena cava avoiding any mechanical damage to the atrium, and pacing stimulation is delivered through the catheter. An electrophysiological study is performed with programmed stimulation including constant pacing, burst pacing, and up to triple extrastimuli pacing. Under a spontaneous or pacing rhythm, the optical mapping recorded the action potential duration, activation map, conduction velocity, and Ca2+ transient individually in the right and left atria. In addition, the programmed stimulation also determines the inducibility of atrial tachyarrhythmias. Precise activation mapping is performed to identify the propagation of the excitation in the atrium during an induced atrial tachyarrhythmia. Optical mapping with a specialized setting enables a thorough electrophysiological evaluation of the atrium in murine pathological models.

This protocol describes the electrophysiological evaluation of murine atria utilizing an optical mapping system with a high temporal and spatial resolution, including dual recordings of the membrane voltage and Ca2+ transient under programmed stimulation through a specialized electrode catheter.

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and ...

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Medicine

Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of metabolic syndrome (MetS)-related heart failure with preserved ejection fraction (HFpEF). The prevalence of MetS-related HFpEF is rising, and atrial cardiomyopathies associated with atrial remodeling and atrial fibrillation are clinically highly relevant as atrial remodeling is an independent predictor of mortality. Studies with isolated single-cell cardiomyocytes are frequently used to corroborate and complement in vivo findings. Circulatory vessel rarefication and interstitial tissue fibrosis pose a potentially limiting factor for the successful single-cell isolation of ACMs from animal models of this disease.
We have addressed this issue by employing a device capable of manually regulating the intraluminal pressure of cardiac cavities during the isolation procedure, substantially increasing the yield of morphologically and functionally intact ACMs. The acquired cells can be used in a variety of different experiments, such as cell culture and functional Calcium imaging (i.e., excitation-contraction-coupling).
We provide the researcher with a step-by-step protocol, a list of optimized solutions, thorough instructions to prepare the necessary equipment, and a comprehensive troubleshooting guide. While the initial implementation of the procedure might be rather difficult, a successful adaptation will allow the reader to perform state-of-the-art ACM isolations in a rat model of MetS-related HFpEF for a broad spectrum of experiments.

Here, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes from a rat model of metabolic syndrome-related heart failure with preserved ejection fraction. A manual regulation of intraluminal pressure of cardiac cavities is implemented to yield functionally intact myocytes suitable for excitation-contraction-coupling studies.

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

Langendorff Retrograde Heart Perfusion: An Ex Vivo Technique to Perfuse Isolated Mouse Heart

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