Dual-Dye Optical Mapping of Hearts from RyR2^R2474S Knock-In Mice of Catecholaminergic Polymorphic Ventricular Tachycardia

This method will help us to reveal the electrophysiological properties and mechanisms of ventricular arrest smears associated with diseases such as catecholaminergic polymorphic ventricular tachycardia. Using this technique, we can obtain the membrane potential and intercellular calcium signals simultaneously under various programmed electrical pacing protocols, which is especially suitable for exploring the underlying mechanisms and dynamics of cardiac arrhythmic disease such as CPVT. To perform this experiment successfully, ensure to have a well perfused heart, proper dye loading, excitation-contraction uncoupling, and careful camera settings.

To begin, set up the optical mapping system and turn on the camera for stable sampling temperature at minus 50 degrees Celsius. Place the harvested mouse heart into the cold CREB solution to slow metabolism and protect the heart. Remove the surrounding tissue of the aorta.

Use a custom made cannulating needle to cannulate it, and fix it with a 4-0 silk suture. Now perfuse the heart with the Langendorff system at a constant speed of 3.5 to 4.0 milliliters per minute. Insert a small plastic tube into the left ventricle to release the congestion of solution in the chamber to avoid over preload and fix the plastic tube in the cannulating needle.

Then place two leads into the perfusate in the bath and switch on the powers for the electrocardiogram amplifier box and electric stimulation controller. Next, initiate the referenced electrocardiogram or ECG software and continuously monitor the ECG. Perform the subsequent steps in the dark when the heart reaches a stable state condition.

Perfuse the blebbistatin CREBs solution mixture constantly into the heart for 10 minutes to uncouple contraction from excitation and avoid contraction artifacts during filming. Then using a red flashlight, examine the heart to confirm the complete cessation of contractions. Perfuse the heart with Rhod-2 AM working solution for 15 minutes in the Langendorff perfusion system after uncoupling excitation contraction.

Maintain oxygen supply during intracellular calcium dye loading. To prevent bubble formation from pluronic F-127, insert a bubble trap into the perfusion system. Dilute 10 microliters of RH 237 stock solution into 50 milliliters of perfusate and load for 10 minutes.

Upon completing the dual dye loading, capture a sequence of photos. Confirm that voltage and calcium signals are adequate for analysis. Turn on the two LEDs for excitation lights and adjust their intensity to a proper range.

Place the heart under the detection device, ensuring it is well illuminated by the two LEDs adjusting the light spot diameter to two centimeters. Set the working distance between the lens and the heart at 10 centimeters to achieve the desired sampling rate and spatial resolution. Open the signal sampling software to simultaneously control the camera and capture voltage and calcium signals.

Start the MyoPacer Field Stimulator and set the pacing pattern to transistor transistor logic with two milliseconds of pacing duration per pulse. Set the initial intensity to 0.3 volts. Position a pair of platinum electrodes attached to the epicardial of the left ventricle apex.

Apply 30 consecutive 10 hertz S1 stimuli to test the heart's diastolic voltage threshold using the ECG recording software. Gradually increase the voltage amplitude until one-to-one capture is achieved. Implement the S1-S1 protocol to measure calcium or action potential alternates and restitution properties.

Pace the heart consecutively starting at a basic cycle length of 100 milliseconds. Decrease the cycle length by 10 milliseconds in each subsequent sequence until it reaches 50 milliseconds. Simultaneously initiate optical mapping prior to stimulation.

To measure the ventricular effective refractory period using the S1-S2 stimulus protocol, start with an S1-S1 pacing cycle length of 100 milliseconds. Couple S2 at 60 milliseconds and decrease with a two millisecond step decrement until S2 fails to capture ectopic QRS complex. For arrhythmia induction, administer perpetual 50 hertz burst pacing and perform the same pacing episode after waiting for a two second interval of resting.

Carefully monitor the electrocardiogram recordings during the continuous high frequency pacing period to promptly begin simultaneous optical mapping recordings when an interesting arrhythmic wave is generated. Proceed to capture images using the electron multiplying charge coupled device camera. In the image acquisition software, press Select Folder, and load images to begin the semi-automatic massive video data analysis process.

Input the correct sampling parameters for the analysis. Manually set the image threshold and select the region of interest. Apply a three by three pixel Gaussian spatial filter, a Savitzky-Golay filter, and a top hat baseline correction.

Then press Process Images to remove the baseline and calculate electrophysiological parameters like APD-80 and CATD-50. Set the initiation time of action potential duration at the peak and the terminal point at 80%repolarization for calculating APD-80. Similarly define the start time of calcium transient duration as the peak with the terminal point being 80%relaxation.

Typical traces in heat maps of APD-80 and CATD-80 are shown. Isoproterenol shortens APD-80 and wild type and catecholaminergic polymorphic ventricular tachycardia or CPVT mice, but no difference was found after isoproterenol challenge. CATD-80 and CPVT mice was longer than in wild type after the isoproterenol challenge while there was no significance before treatment.

According to the voltage signals, the wild type and CPVT hearts possessed the same conduction ability across the epicardium at baseline and after isoproterenol intervention. Heat maps demonstrated that CPVT mice have the same conduction ability as the wild-type mice before and after the isoproterenol challenge. Calcium amplitude alternates analysis showed that the calcium signals in wild-type hearts stayed stable at baseline during consecutive S1-S1 pacing at 14.29 and 16.67 hertz, while the CPVT hearts showed frequency dependent alternates.

After the isoproterenol challenge, CPVT hearts exhibited frequency dependent alternates and calcium signal during S1-S1 pacing, while wild-type hearts were not influenced. Tachy arrhythmia analysis suggested that both wild type and CPVT hearts exhibit normal conduction during 50 hertz burst pacing at baseline. After profusion with isoproterenol, CPVT hearts showed high frequency rotors after 50 hertz burst pacing, while wild-type hearts maintained normal conduction.

Following this procedure, adogen types and wild-type mice are used to illustrate the electro physiological and functional properties in these models or in the invention of pharmaceuticals. Optical mapping is a powerful tool for study cardiac arrhythmias, however it cannot be used clinically due to limitation in fluorescent dye under the excitation contraction uncoupling. With the development of fluorescent proper for different target molecule under the development of high revolution computation technology, the cardiac optic mapping technique is bound to achieve only applications.