Using the protocol as a biophysical effect of different optogenetic actuators on cardiomyocyte activity can be tested to aid in the development of optogenetic experiments in cardiac tissue and whole hearts. By combining the patch clamp technique with sarcomere tracking and carbon fiber assisted force measurements, we can study the effects of GtACR1 photoactivation on the electrics and mechanics of ventricular cardiomyocytes. Optogenetic inhibition can potentially be used for optical defibrillation.
GtACR1 is an optogenetic tool that enables the silencing of cardiac activity. This technique is fully applicable to other research fields such as smooth and single-cell muscle cell studies. Although this protocol can't be used for high throughput screening, it provides a means for the comprehensive analysis of cardiomyocyte electrical and mechanical function.
So as the saying goes, a picture says more than a thousand words. How much more information can we get across in video? Some of our tools and techniques involved in single myocyte stretching and in probe preparation are highly intricate and it is much easier to convey them in a video compared to a description.
After the blood has been washed out of the saline perfused heart from a nine to 10-week-old New Zealand white rabbit, switch the perfusate to a low-calcium, high-potassium solution and perfuse the heart for two more minutes after the heart stops beating. Perfuse the enzyme solution, start recirculating the enzyme solution back into the reservoir after two minutes of digestion, and decrease the speed to 16 milliliters per minute after five minutes of digestion. Once the tissue appears soft after 40-50 minutes of digestion, cut the heart off the cannula and immediately place the heart in blocking solution.
Add blocking solution until the tissue is fully covered. Cut off the right ventricle and septum and separate the left ventricle. Remove the papillary muscles and use fine forceps and a pipette to gently pull apart the tissue to release the cells by mechanical dissociation.
Filter the cell suspension through a mesh with one millimeter squared pores and sediment the cells by centrifugation. Then resuspend the cardiomyocyte pellet in fresh blocking solution. For a patch clamp experiment, coat autoclaved coverslips in a Petri dish with 100 micrograms per milliliter of laminin immediately before cell culture.
For carbon fiber experiment, coat Petri dishes with 0.12 grams per milliliter of Poly-HEMA in a 95-to-five ethanol-to-water solution. Ten to 15 minutes after resuspending the cardiomyocytes, remove the supernatant and resuspend the cells in culture medium. After counting, seed the cells onto a coverslip in a Petri dish at a target density of 1.75 times 10 to the four cells per milliliter.
Incubate the cultures for three to four hours at 37 degrees Celsius and 5%carbon dioxide. At the end of the incubation, replace the medium from the coverslip cultures with fresh medium containing adenovirus type five coding for GtACR1-eGFP at a multiplicity of infection of 75. Return the cultures to the incubator for 48 hours.
To perform a patch clamp experiment, use a micropipette puller to pull 1.7 to 2.5 megaohm patch pipettes from soda lime glass capillaries and initialize the data acquisition software. Place a coverslip with cells into the measuring chamber containing external solution and select the cardiomyocyte based on its eGFP fluorescence. Next, fill a patch pipette with internal solution and attach the pipette to the pipette holder inserting the silver chloride coated silver recording wire into the internal solution.
Set the membrane test to apply 10 millivolt pulses for 15 milliseconds with a baseline of zero millivolts. After reaching the cell-attached configuration, switch to whole cell mode with a holding potential of minus 60 millivolts in the data acquisition software. Then gently apply negative pressure to access the whole cell configuration by rupturing the membrane.
A successful rupture will be evidenced by an immediate increase in the measured capacitance. Record photoactivation protocols in voltage clamp mode at minus 74 millivolts with light pulses of 300 milliseconds or action potentials in current clamp mode at zero picoampere. To produce the carbon fibers, first mount the pipette onto the pipette holders of the puller.
Pull the glass capillaries into two pipettes with a total tapered length of approximately 11 millimeters and a final inner diameter of about 30 micrometers. Next, use a stereo microscope to align one capillary in the orientation circle and bend it by up to 45 degrees by pushing down on the tip of the capillary with a bender. Heat up the filament until the capillary maintains the 45 degree angle even after the bender is removed.
After bending the capillary, use fine forceps equipped with soft tubing to take one carbon fiber out of the tube and fit it into the fine tip of the capillary. Push the fiber in the capillary up to the bend. Cut the carbon fibers so they project two milliliters from the tip of the capillary and use cyanoacrylate glue to fix the fibers to the front section of each capillary.
To calibrate the fibers, attach one capillary to a holder controlled by a micromanipulator and a piezo motor. Move the capillary towards the sensor and align it relative to the sensor. Place the tip of the fiber in contact with the force sensor without producing any force.
The total movement of the piezo motor is 60 micrometers. Move the piezo motor in six 10 micrometer steps toward the force sensor. The sensor has a sensitivity of 0.05 millinewtons per volt and a force range of zero to 0.5 millinewtons.
Read out the measured voltage for each step and remove the carbon fiber from the sensor. To record the force of contracting cardiomyocytes, coat the surface of a coverglass with Poly-HEMA and place it in the measuring chamber. Fill the chamber with external bath solution.
Attach both carbon fiber loaded capillaries to the stage micromanipulator and align them at an angle so that the carbon fibers are near horizontal to the surface of the measuring chamber. To check if the fibers are correctly aligned, focus on the surface of the measuring chamber, lower the first fiber, and move the fiber horizontally. The tip of the fiber should be sliding on the surface of the measuring chamber.
Follow the same procedure for the second fiber. With the lights off, add a few drops of cultured cell suspension to the chamber and apply short green light pulses to select cells that contract in response to the green light stimulation. To attach the first fiber, gently compress the cell by lowering the fiber then release the pressure.
Attach the second fiber parallel to the first one at the other end of the cardiomyocyte in order to have a maximum number of sarcomeres between the two fibers. After both fibers are attached, lift the fibers so that the cell is no longer in contact with the chamber surface. Bring the sarcomeres into focus, set the sarcomere length tracking window between the fibers, and use the edge detection module to track the fiber bending.
Set the detection areas with the red and green windows and define a threshold at the first derivative of the light intensity trace. Optically pace the cell while tracking sarcomere length and fiber bending. In this case, we paced at 0.25 hertz.
After recording at least 15 optically elicited contractions, field stimulate the cell electrically. Find the threshold for eliciting the contractions and apply 1.5 times the threshold voltage for electrical pacing. For an inhibition protocol, apply electrical stimuli to elicit contractions and then expose the cell to a sustained light pulse at various light intensities.
Record at least 15 contractions after light-induced inhibition. GtACR1 is expressed in cultured rabbit cardiomyocytes. GtACR1 photoactivation at a light intensity of four milliwatts per millimeter squared for 300 milliseconds results in large inward-directed currents at minus 74 millivolts with a measured peak current of 245 picoampere.
In this representative experiment, action potentials were triggered either electrically with current injections 1.5 times the threshold or optically with 10 millisecond light pulses. Optically paced cardiomyocytes demonstrated a slower action potential onset. Electrically triggered action potentials were inhibited under sustained light.
Higher current injections and 1.5 times the threshold during the sustained light application also do not elicit action potentials. The cardiomyocyte generated a contraction force of 232 micronewtons per millimeter squared upon electrical pacing and 261 micronewtons per millimeter squared following optical pacing. Prolonged green light pulses inhibited the contractions with the post-inhibition reoccurring contractions generating a lower contractile force in keeping with the diastolic calcium loss from rabbit cardiomyocytes.
We recommend practicing the techniques before conducting an experiment, particularly as positioning the microprobes in the dark can be challenging. is very important when analyzing cardiomyocyte contractility as it can affect force production and relaxation. Various afterloads can also be applied for isotonic and auxotonic contraction analysis.
These techniques allow characterization of the biophysical effects of activating newly developed optogenetic actuators in cardiomyocytes which is crucial for selecting the most suitable optogenetic tool for each experiment. Please be aware that adenovirus transduction and all of the steps following the transduction should be performed under biosafety level II conditions.
