This approach detects changes in contractile function during the development of heart failure. The functional response in myocytes to neurohormones and potential therapeutic agents can also be screened. Positioning the myocyte may take practice but is essential in obtaining consistent sarcomere length imaging.
Demonstrating the procedure is Margaret Westfall, associate professor and principal investigator of the laboratory. Before starting the experiment, preheat a gel pack and media in an incubator at 37 degrees Celsius for 30 minutes. Turn on the contractile function platform components as mentioned in the manuscript and assemble tubing into the peristaltic pump.
Then, transfer the preheated gel pack into the tube holder and turn on the vacuum in the power to the cell stimulator. Place a 50 milliliter tube with media in the insulated tube holder to begin perfusion at 0.5 milliliters per minute through the peristaltic pump tubing. Next, add two milliliters of preheated media to a small weigh boat and transfer one cover slip containing myocytes from the stimulation chamber to the weigh boat.
Return the stimulation chamber to the incubator at 37 degrees Celsius and 5%carbon dioxide. Remove the cover slip from the weigh boat and gently wipe the underside before transferring it to a freshly greased cover slip chamber and adding a drop of media onto the cover slip. Place a platinum electrode mount over the cover slip, then lay a new cover slip over the top with forceps and place a top mount over the cover slip sandwich.
Install two or four number zero panhead screws to finish assembling the chamber. In the peristaltic pump when the media is present throughout the tubing, attach the tubing to the preheater and the preheater to the cover slip chamber. Then, begin perfusion at 0.5 milliliters per minute and place a tissue wipe below the chamber to ensure no leaks.
After placing the cover slip chamber in the stage adapter of the microscope, turn on the heating system and equilibriate the assembly for five to 10 minutes to achieve a constant media temperature of 37 degrees Celsius in the center of the chamber. Observe the perfused media being collected at the opposite end of the chamber with tubing connected to a vacuum system. During the equilibration time, visualize myocytes with the 40x water immersion objective on the microscope.
To activate the pacing stimulator, set the voltage to 35 to 40 volts and adjust the stimulation frequency to 0.2 hertz to optimize contractile function and myocyte survival. To collect sarcomere shortening traces, open the software on the computer and select okay, file and new tabs. Prepare a screen template by selecting traces, edit user limits and sarcomere length.
Record sarcomere length traces with the file and new tabs, identify a contracting myocyte and position the myocyte along the camera's longitudinal axis so the striation pattern is vertical. Use the computer mouse to place the region of interest or ROI box over the myocyte. Select collect and start to record contractile function.
Record shortening for 60 seconds from each myocyte at low stimulation frequencies of 0.5 hertz. Record sarcomere shortening from five to 10 cells per cover slip. To measure shortening over a range of frequencies, place the myocytes at each frequency to obtain steady state shortening prior to recording.
Double the perfusion rate and begin recording 15 to 20 seconds after stimulation at a new frequency in the range of 0.2 to two hertz and record no more than two myocytes per cover slip for shortening across the given range of stimulation frequencies. Record at least seven contractions per cell to obtain reliable signal averaged data. Select the file, choose a recorded trace and then select open.
Select tab one of the base trace and then set the yellow panel at the top of the trace to obtain sarc length. Prepare an analysis template with operations monotonic transient analysis options and TTL event mark in the definition of T Zero box in the necessary options from the menu. Save these analysis options by selecting templates and save analysis template with an identifier.
Load the analysis template before analyzing each trace. To analyze the data, select marks, gate, add transient then convert from event mark and analysis range. Set the time range from minus 0.01 to 1.20 seconds for myocytes paced at 0.2 hertz.
Select a shorter time range for myocytes stimulated at higher frequencies. Select operations and average events to produce a signal averaged recording below the original base trace. Next, select tab one of the signal averaged trace and then go to marks on the upper menu followed by add transient, choose operations and monotonic transient analysis to display the signal averaged values for baseline sarcomere parameters in the signal averaged display panel.
Select export monotonic transient analysis and clipboard current and transfer transient sarcomere analysis to a spreadsheet for the composite analysis of multiple myocytes. To copy the signal averaged trace, select export, current trace, then clipboard and options tabs in order and set the decimal places to five. Choose tabs delimiter and click okay.
Pace the signal averaged traces for each myocyte recording into a second spreadsheet. The study showed that the pressure overload or PO, reduced the contractile function of the myocytes when compared to the sham group. However, four days after the gene transfer of cardiac troponin IT 144D or cTnIT144D, the contractile ability in the PO myocytes returned towards sham levels, indicating myocyte function could be restored.
In the initial studies, the gene transfer of cTnIT144D enhanced peak shortening and elevated diastolic calcium levels compared to CTnI. Fine adjustments in positioning of the cardiac myocyte are often needed to obtain clear shortening traces. This protocol can also be performed on cardiac myocytes loaded with calcium sensitive fluorescent dyes such as Fura-2 AM to detect calcium transients in addition to shortening.
