This protocol can be used to assess the impact of genetic mutations on muscle fiber function. In addition, it can be used for drug screening studies aimed at improving muscle health. This technique can be used to isolate and measure contractility in a large number of muscle fibers in a relatively short period of time without the need for advanced training.
The hydrogels'composition can be changed in order to attest the effect of environmental cues in both healthy and diseased muscle. The most challenging part of the procedure is the initial dissection of the muscle. If enough care is not taken, damage to the muscle can lead to a decrease in the viability of the muscle fibers.
Demonstrating the procedure will be Leander Vonk, a research technician, and Osman Esen, a PhD candidate in my laboratory. Start by cutting the lower hind leg of the euthanized mouse just above the ankle. Make an incision on the skin on the dorsal side of the foot towards the toes.
Carefully peel the skin towards the toes taking care not to damage the muscle. Place the dissected foot in a seal guard dish containing 10 milliliters of pre-warmed dissection medium at 37 degrees Celsius. Pin the foot through the skin still attached to the toes.
Pin the lower leg beyond the ankle and carefully excise the connective tissue on top of the muscle. Cut the tendon at the heel and lift the muscle. Cut alongside and underneath the muscle through the connective tissue until the toe tendons are exposed.
Cut the tendons when the half of the length of three tendons are exposed. Release the muscle from the foot and pin the tendons of the FDB muscles. Remove any remaining connective tissue from the muscle.
Transfer the clean muscle tissue to a tube containing pre-warmed dissection medium. Use a serological pipette to transfer the brevis muscle to a tube containing the muscle digestion medium. Place the tissue in an incubator at 37 degrees Celsius under 5%carbon dioxide for 80 minutes.
Transfer the muscle to a 15 milliliter tube containing three milliliters of dissection medium. Using prepared trituration tips, pipette the muscle going from the largest to the smallest and continue trituration until the muscle fibers have come off the tendon. The tendons can be removed with a P200 tip.
Transfer the dissociated muscle fibers into a 15 milliliter tube containing 10 milliliters of dissection medium. Allow the fibers to settle in the incubator for 20 minutes until a pellet is formed. Carefully pipette all the medium from the top of the fiber pellet.
On the ice, resuspend the cells in 875 microliters of cell mix per muscle. Add 125 microliters of matrix cell mix into tubes containing 125 microliters of cell suspension aliquot. Mix by gentle pipetting, avoiding the formation of bubbles.
Transfer the final mix immediately into a well. Check the viability of the muscle fibers under the microscope. Place the gels in an incubator for 30 to 45 minutes to promote solidification.
Once done, carefully add 500 microliters of culture medium into each well. Turn on the optics-based contractile measurement system, the fluorescent lamp, the electrical cell pacer, and the computer. Set the electrical stimulator to 1.0 hertz at 10 volts with a pulse duration of five milliseconds to stimulate the isolated muscle fibers.
Insert the plate into the measurement system. Connect the pacer to the insert and insert it into the culture plate. Open the program IonWizard.
Click OK.Open a new file by clicking on the File icon, then click New. Select Collect Experiment to check if the program is on the correct experiment skeletal sarcomere. To change the experiment, click on the desired experiment and then press Add.
Apply the settings SARC 20X, average lines, single FFT, 250 hertz sampling rate, and an acquisition time of 10 seconds. Change the measurement system temperature to 25 degrees Celsius. Click on the open cell finder to create a new screen popup.
Select Plate Type and Active Wells. Focus the fibers by adjusting the focus slider bar. Enable the pacing to induce electrical stimulation and observe fiber twitching.
Keeping the sarcomeres in focus, ensure the measurement area is fixated on the fiber end such that the sarcomeres run vertically. When the sarcomeres are focused, a single peak is visible in the toolbar which will move to the right upon contraction. Measure the fibers on the measuring system.
Clicks Start to begin the experiment. Press the Q key to start measuring 10 contraction transients. If more than four transients look noise free, accept the measurement by pressing the Z key.
If the transients contain too much noise, reject the measurements. Press Stop to close the cell finder window once the experiment concludes. Save the file and create a new file.
Open the program CytoSolver desktop, then click on Import and select the files to be analyzed. Once analysis is complete, identify the blue, red, and gray peaks. Click on Export.
Select the boxes titled Average to Transient Data and export to Excel. A higher percentage of usable contractile muscle measurements was obtained in the 3D fibrin hydrogel since it prevented lateral movements and other influencing factors. Fiber embedding had no significant effect on the maximum contraction speed or the sarcomere shortening.
Reduced contraction in the pure basement matrix was observed likely due to gel stiffness or increased cell matrix interaction. Similarly, basement matrix embedding also reduced sarcomere shortening compared to the fibrin hydrogel. It's important to remember that the isolated muscle fibers are quite fragile and extra care should be taken not to damage them during pipetting.
After the isolation procedure, methods such as immunostaining, protein and RNA isolation may also be performed. The development of this technique has opened up new possibilities to study mature muscle fiber function in a high throughput manner.
