The protocol presents Drosophila models where the heart function can be characterized and controlled using light. This enables researchers to study human heart diseases in Drosophila using intact animals. This method can reliably achieve non-invasive OCT imaging and optogenetic control of Drosophila heart function.
It has high efficiency and quality. Optogenetic pacing can potentially be an alternative to electrical pacing as therapy to treat arrhythmic disorders. OCT imaging and optogenetic pacing can be extended to other model systems like heart organoids, zebra fish and embryonic throat and heart.
To begin, combine five hand GAL4 over TM6 double-tubby virgin female and two to three male flies from UAS Ops and Stocks per vial. On the next day, prepare semi defined food according to the instructions of the Bloomington Drosophila Stock Center by adding 5.14 grams per 100 milliliter sucrose into a container on a hot plate. Then cool it to 60 degrees Celsius with constant stirring.
Next, prepare narrow fly vials by adding 50 microliters of 100 millimolar all-trans retinal ethanol solution to each vial. Use a serological pipette to dispose of five milliliters of fly food per narrow fly vile. After vortexing at maximum speed for 10 seconds, plug these vials and wrap them in the dark fabric to protect them from light.
The next day, transfer the flies steadily laying eggs to the vials with all-trans retinal ethanol containing food. Protect the racks with vials from light. After 24 to 48 hours, depending on the number of eggs laid, discard the parents to prevent vial overpopulation.
Next, collect non-tubby progeny for heart imaging. Pick UAS option hand GAL4 larva or pupa from the vial, put it on a tissue, and gently wipe off the media from the body surface using a painting brush. Prepare the microscope slide with a small piece of double-sided tape in the middle.
Next, gently place the larva or pupa on the tape surface with the dorsal side up and perpendicular to the long side of the slide using a brush or fine tweezers. Apply gentle pressure to attach the larva or pupa to the tape's surface. Now, set up the slide on the imaging stage with the larva or pupa facing down and turn on the optical coherence tomography or OCT light source by laser control software.
Open the custom written spectral domain OCT control software. Then, click on the preview window. Then, set the scan parameters in the spectral domain OCT software.
Use micro manipulators to control the sample stage to bring the fly heart into focus. Adjust the focal position to minimize light reflection from the fly cuticle surface. Also, consider applying mineral oil on the larva or pupa surface to minimize reflection.
Next, set the scan parameters for M mode OCT image acquisition and acquire five sets of control data without red light stimulation pulses to calculate resting heart rate. Design the light pulse for the pacing stimulation in the custom OCT control software. For this, click on the settings tabs and add the designed light pulse sequences to control pulse frequency, pulse width, stimulation duration and waiting time according to different stimulation protocols.
Then open the light controller software to generate red light pulses. Choose the pulse mode in mode selection. Double click the figure for the pulse profile settings, and choose follower mode.
Keep the off intensity at zero and set the on intensity percentage upon calculating the actual power density. Acquire M mode videos of the beating Drosophila heart with light stimulation by clicking on Acquire in the OCT Control software. Record flashes of red light down the fly heart during imaging acquisition.
Note that different pacing settings are required to control the fly heart function with red-shifted channelrhodopsin and NPHR fly models. Open the custom developed fly heart segmentation software and click on select file. Then select the file to be analyzed in the graphical user interface.
Enter both the vertical and horizontal boundaries of the heart region in pixels in the top text boxes. Click on resize. Using the slider on the bottom, ensure that the entire heart region is visible and that it fills up the whole box for the entire collection.
After clicking the predict tab, the program will go through every slice in the collection and select the heart region in approximately three minutes. Once the prediction is completed, click on the HR plot to display a plot of the heart area over time in a new window. Select the correct peak or valley areas, choose the pulse and then HR tabs to generate a final figure.
The functional parameters will be simultaneously saved in the CSV files. The tissue specificity of the hand GAL4 driver was verified by imaging green fluorescent protein expression. The typical OCT images of larval and pupal body cross section are shown here.
To mimic different heart conditions, four types of light pulses were designed. A single pulse lasting 10 seconds after five seconds of waiting time, generated restorable heart arrest. For the heart pacing at frequencies lower than the resting heart rate, two light pulse sequences with pacing frequencies equal to half of the resting heart rate, and one fourth of the resting heart rate lasting eight seconds with a waiting time of six seconds in-between were used.
The stimulation pattern to increase the heart rate due to red-shifted channelrhodopsin activation consisted of three sequences of light pulses. The reduced heart contraction frequency following the light signals resulted in a slower heart rate that mimics in larva and pupa. A series of three light pulse trains at different stimulation frequencies was applied on larvae and pupae hearts clearly showed increased heart rate following the light pulses.
The preparation selection of correct progeny, mounting of the specimen, and imaging procedure are essential. We hope to transfer the optogenetic pacing technology to larger animal models, to study mammalian heart diseases and to explore new therapeutic approaches to treat arrhythmia.
