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 × 2 mm or 10 mm x 10 mm, with a 100 × 100 resolution (20 µm/pixel or 100 µm/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.


Introduction
The heart consists of 4 chambers in mammals. The upper two chambers are atria, and the lower ones are ventricles. Ventricles work as a pump to eject blood to the systemic or pulmonary circulation. Atria receive blood returning from the systemic or pulmonary veins, and assist in transporting blood into the ventricles to obtain an efficient cardiac pump function. From an electrophysiological aspect, the important function of the atria is to regulate the heart rhythm. The electrical signals originate from the sinus node located at the junction between the superior vena cava (SVC) and right atrium (RA), then propagate to the RA and the left atrium (LA), and conduct to the ventricle through the atrioventricular node and His-Purkinje conduction system. Arrhythmias, which are heart rhythm disorders, are classified into atrial and ventricular according to their origin. Atrial fibrillation (AF) is the most common sustained form of an arrhythmia, characterized by a random and rapid excitation of the atria. Recent genetic analyses and genomewide association studies (GWAS) have shown the association between AF and genetic mutations or monopolymorphisms 8. Puncture a 24-gauge indwelling needle (outer diameter: 0.7 mm) into the left ventricular (LV) cavity through the ventricular apex to avoid any damage to the atrium, and remove the inner needle leaving the external cannula in the LV. 9. Introduce a 1-French size custom made electrode catheter through the PE tube in the SVC to perform electrical stimulation in the RA. If needed, advance the catheter into the right ventricle (RV) for ventricular pacing. 10. Insert a pin electrode into the ventricular apex to continuously record the bipolar electrocardiograms (ECG) between the pin electrode and cannulation needle in the ascending aorta (Figure 1c). 11. Continue monitoring the ECG and perfusion pressure throughout the entire study. 12. Turn off the light, and perform the following experiment in a dark room. 13. Go to step 2.5. for a single recording of the membrane voltage or 2.6. for a dual recording of the membrane voltage and Ca 2+ transient.

5.
Staining for a single recording of the membrane voltage 1. Maintain the perfusion solution heated at 37 ˚C during this staining protocol for a single recording of the membrane voltage.

Representative Results
The optical mapping experiments are performed using the devices as shown in Figure 1a. The schema of the optical system is illustrated in Figure 1b. The system provides a high-resolution analysis of the membrane potential and Ca 2+ transient in the atrium.
As depicted in Figure 1c and d, the isolated heart is positioned in a temperature-controlled chamber. A 1-French size electrode catheter is placed in the RA through a PE tube inserted into the SVC, and another indwelling PE tube is inserted into the LV cavity from the LV apex to avoid excessive pressure on the heart chamber. For a simultaneous ECG recording, the cathode (ECG lead [-]) is connected to the cannulation needle, and the anode (ECG lead [+]) is connected to the pin electrode inserted into the ventricular apex. The feature of this assembly is to avoid any mechanical damage to the atria.
For the evaluation of the membrane voltage, fluorescence using di-4-ANEPPS is recorded with up to a 10,000 frames/s sampling rate (Figure 2). The activation map is obtained during constant pacing delivered from the RA. The fine mapping enables the analysis of the detailed conduction pattern in small murine atria. pacing from the RA. We reduce the sampling rate of the mapping to 1000 frames/s to obtain a sufficient signal/noise ratio of the Rhod2AM signal with the present setting. However, the system provides a sufficient quality for analyzing the relationship between the action potential and Ca 2+ transient.
We then perform an induction of an atrial tachyarrhythmia with programmed stimulation using mice under a pathological condition. Figure  4 exhibits an optical recording of an induced AT in a murine atrium stained with di-4-ANEPPS. The mouse undergoes a transverse aortic constriction procedure to apply a pressure overload to the atrium 10 days before the experiment 6 . We induce AT by triple extrastimuli pacing (120/100/100/80), and observe the propagation of the reentrant circuit.   Supplementary movie 1. Representative movie of the membrane potential in the atrium using staining with di-4-ANEPPS. Please click here to download this file.

Supplementary movie 2.
Representative movies of the activation mapping during atrial tachycardia and sinus rhythm. Please click here to download this file.

Discussion
Optical mapping is a well-established maneuver for studying the cardiac electrophysiology 7 , and is a quite useful tool to assess not only ventricular arrhythmias 8,9 , but also atrial ones 10,11 . Simultaneous mapping of the transmembrane potential and Ca 2+ transients is useful for understanding the underlying mechanisms of arrhythmias in relation to heart failure and other heart diseases 12,13 . When comparing the other electrophysiological assessment methods, such as those using a single cell or cell sheet, one of the absolute superiorities of optical mapping in the perfused heart is the assessment of the conduction pattern in the intact atrium and ventricle, not only during sinus rhythm but also during induced arrhythmias , our method has the advantage of assessing the murine atrium by making some modifications to it. First, we pursued to obtain a high spatial and temporal resolution of up to 0.1 ms/frame and 20 µm/pixel, and this high-resolution mapping contributed to a more precise measurement of the conduction velocity and propagation pattern in the murine atrium. Second, to avoid any unnecessary mechanical damage or stretch of the atrium, which could alter the electrophysiological properties 16,17 , an indwelling needle is inserted directly into the LV to reduce the intra-chamber pressure, instead of inserting it through the LA as performed in the previous study 15 . Furthermore, the pacing stimulus is delivered through a custom made 1-French size electrode catheter placed in the RA, but not by a needle electrode, which might injure the atrium. Any pins are avoided in fixing the atrial appendage, which were used in the past study 15 . Third, in terms of the assessment of the underlying mechanism of the arrhythmias, a programmed stimulation protocol to induce atrial tachyarrhythmias is crucial 18,19 . We perform programmed stimulation identical to that in clinical electrophysiological studies, including burst pacing and up to triple extrastimuli pacing, with a modification of the pacing interval for the mouse heart. Thus, in addition to the baseline measurement parameters, the protocol could evaluate the inducibility of the AT. When needed, the inducibility of the AT is assessed with the administration of isoproterenol or other drugs. In our experience, the wild-type mice hardly show any ATs even after a full stimulation protocol. Thus, the inducibility of AT should be important information for evaluating the contribution of several pathological conditions such as genetic mutations, surgical procedures, and the administration of drugs 11 . Those modifications could optimize the precise electrophysiological assessment in the intact murine atrium.
This method also has some limitations. First, using a maximum spatial resolution with a 5X objective lens, the field of view (FOV) is limited to a part of the atrium (i.e. only the left atrial appendage as shown in Figure 2a). For obtaining the larger FOV of the atrium, a 1.6X objective lens is sometimes preferable (Figure 2b). Second, without fixing the atrium with pins, sometimes it is difficult to measure the atrial conduction properties correctly, because the atrial surface is curved. So, we placed the cover glass on its surface to flatten it instead of fixing it by pins. This method is also beneficial for preventing motion artifact from vibrations of the solution. Third, with our method, it is quite difficult to obtain the entire FOV of it, so, to use the anterior and posterior view properly is more important in our approach than in the other approach as shown in Figure 2. The advantage of the anterior view would be the clear observation of reentry in the case of pathological conditions, especially in the appendage (Figure 4). On the other hand, the posterior view has an advantage of obtaining a good view of the atrial posterior wall, and might be a detailed recording of triggering activity from the myocardial sleeve. When it is difficult to obtain an appropriate view and to flatten its curved surface with our method, the atrium can be fixed with minimal tension by pins.
With our method, there are 3 possible problems, failure of staining, pacing, and arrhythmia induction. For failure of staining, if no or slight fluorescence is observed, you should check whether the optical mapping apparatus is correctly assembled, and whether the reagent is appropriately stored and used. The condition of the perfusion solution is also crucial, which also can affect the electrophysiological properties of the heart itself, so, the condition of solution including the pH, temperature, and whether there was enough aeration has to be strictly monitored. It is also important to avoid any air emboli in the heart. For pacing failure, if the pacing stimuli cannot excite the atrium, researchers should check whether the wiring is correct using a circuit tester. When the pacing stimuli are correctly outputted, the problem is the contact of the electrode with the tissue. Repositioning of the electrodes can resolve the problem, and our approach using the pacing catheter makes it easy. For difficulty in arrhythmia induction, RV pacing can be used for the induction of an AT in some limited cases. Using a quadripolar electrode catheter of which the distal two electrodes and proximal electrodes can be located in the RV and RA, respectively, it is easy to change the pacing site from the RA to the RV. This catheter is also useful for shifting the ventricular excitation when a simultaneous ventricular activation signal masks the atrial excitation signal. This method will contribute to assessing the genotype-phenotype interactions in the AF related genes newly found by the novel studies such as GWAS, especially for the genes with which the investigation failed to show them by other approaches. With the progress of devices and techniques, the electrophysiological properties of the pulmonary vein sleeve, which is the important source of AF 20 , can be assessed in the intact heart with this approach.

Disclosures
The authors have nothing to disclose.