The overall goal of this procedure is to directly visualize and measure the characteristics of calcium sparks in intact skeletal muscle fibers. This is accomplished by first isolating intact single flexor digitorum brevis muscle fibers from a mouse. Next, the muscle fiber is loaded with flu oh 4:00 AM calcium sensitive dye to detect the calcium sparks.
The final step is to record the sparks and analyze the data. Ultimately, confocal microscopy based imaging is used to detect calcium transient induced by transient osmotic stress. This method can help answering the key question in the field of muscle physiology concerning the mechanism that leads to the skeletal muscle dystrophy and the muscle aging process.
So we will have two graduate students, Christine GOPer, and to demonstrate the healthy intake FDB fiber isolation and the senior research scientist, Dr.Park, to demonstrate the osmotic stress perfusion and the data analysis. Begin the setup by positioning a three axis micro manipulator holding a two channel perfusion system. The perfusion system is made with disposable lure syringe barrels with attached three-way lure.
Lock stopcocks load one channel of the perfusion system with isotonic tyro solution and load the other channel with hypotonic tyro solution. After euthanizing a mouse, use heavy dissecting scissors to remove the foot by cutting through the leg above the ankle joint. Place the foot in a dissection chamber filled with minimal calcium tyro solution.
Pin the foot to the chamber with the planter surface facing up. Dissect the flexor digitorum brevis or FDB muscle by exposing and cutting the tendon. Then gently pull up on the tendon to separate the muscle from the surrounding tissue.
Find where the distal tendon branches into individual digits in the deep layers of the muscle and cut the distal tendon at that location. Grasp the tendons with forceps and transfer the FDB muscle into a tube with a thawed aliquot of collagenase digestion solution Prewarm to 37 degrees Celsius. Incubate the tube containing the FDB upright on an orbital shaker at 37 degrees Celsius for 60 to 90 minutes with a speed of 160 RPM after incubation transferred the digested FDB into a tube with 700 microliters of isotonic tyro solution.
Next, use a clean razor blade to remove the tips of three or four, 200 microliter micro pipettes to create micro pipette tips of gradually decreasing diameter with an uncut 200 microliter pipette tip as the smallest diameter. Now starting with the largest diameter micro pipette tip, draw the FDB muscle through the micro pipette tip. Repeat this action several times.
Then switch to the micro pipette with the next smaller diameter tip and draw the muscle several times through that tip. Broken fibers cannot be used for this protocol, so it is important not to over or under digest the fibers and be sure not to force them through too small and opening in the micro pipette tips. Continue through the series until the smallest diameter tip has been used to avoid damaging fibers.
Each tip should be just large enough to allow the fiber bundle to pass through without sticking. This process takes approximately 10 minutes and ends when individual FDB fibers are visible. Next, after gently tapping and resus suspending the FDB fibers in the tube, use a cut 200 microliter micro pipette to transfer 70 microliters into the center of a 35 millimeter delta TPG dish containing one milliliter of isotonic tyro solution.
Then use a dissecting microscope to determine the number of intact single fibers in the dish. Add additional aliquots of FDB fibers if needed to obtain three to four intact fibers on the dish. The remaining isolated muscle fibers can be stored at four degrees Celsius for use within six hours.
For the di loading transfer 500 microliters of isotonic tyro solution from the dish with plated FDB fibers into a tube with 10 microliters of flu oh 4:00 AM stock. After mixing, transfer it back to the dish for a final flu oh 4:00 AM concentration of 10 micromolar. Allow the muscle fibers to download for 60 minutes at room temperature in the dark after an hour.
Carefully remove 500 microliters of flu oh 4:00 AM containing isotonic tyro solution in three batches using an uncut 200 microliter micro pipette tip. Then add an equal volume of fresh isotonic tyro solution. Repeat this wash three more times.
Transfer the dish to the confocal microscope and select an intact fiber with clear striations and a smooth Sarco leal membrane for experimentation. Use the micro manipulator controls to position the tip of the perfusion system, 400 to 500 microns away from the target muscle fiber and off center of the muscle fiber. Begin the flow of the isotonic Tyro solution to ensure that the FDB fiber stays in place.
If the fiber does not stay in place, locate another fiber on the dish and reposition the tip of the perfusion system using a 40 x oil immersion objective. Excite the flu oh 4:00 AM with an argon laser and record the emission signals. Collect a baseline for the osmotic stress experiments by perfusing the fibers with isotonic tyro solution for 60 seconds.
Then switch to hypotonic Tyro solution for 100 seconds to induce swelling, followed by a return to isotonic Tyro solution for at least 10 minutes. Generally, only one osmotic shock is applied to a single intact fiber in one delta TPG dish. Throughout the osmotic stress experiment.
Record the spatial localization of calcium sparks as described in the text protocol. Then find a new fiber on a new dish and repeat the osmotic shock protocol to induce calcium transient. Collect a large number of XT line scan traces to evaluate the morphology and kinetics of individual calcium spark parameters.
A custom developed algorithm is useful for analysis requiring manual identification of regions of interest in some muscle disease models. Alternatively, the publicly available spark master in image J can be used to define the kinetic properties of calcium sparks and their spatial distribution in skeletal muscle. A skeletal FDB muscle fiber was first treated with isotonic tyro solution, then treated with hypotonic tyro solution and then to treatment with isotonic tyro solution.
A histogram of the spark episodes observed with XY imaging is shown here as the muscle fiber shrinks back to the original volume. A robust calcium spark response is observed directly under the sarcolemma of the muscle fiber. The confocal line skin shown by the red dashed line is placed right underneath the Sarco ammal membrane of the muscle fiber.
The osmotic stress evoked discreet calcium sparks, which are presented in line scan or XT mode. Spark analysis with our custom developed spark fit algorithm demonstrates the calcium intensity, which is proportional to the SR calcium release flux, and also demonstrates the kinetics or duration of the sparks. A three-dimensional plot of the flu oh four line scan image shows the osmotic stress evoked sparks in spatial temporal mode and their intensities shown.
Here are line scans or XT images of calcium sparks derived from young healthy FDB muscle fibers, sparks from aged FDB muscle fibers and sparks from young dystrophic MDX mouse FDB muscle fibers. Note the reduced signal that occurs in aged muscle fibers while the young MDX muscle fibers displayed robust calcium sparks. Calcium sparks from wild type EDL muscles are shown here along with TMRE labeling of mitochondria.
The images show transience before and after osmotic pressure changes. Calcium sparks from a myotrophic lateral sclerosis or a LS muscles are shown here. Note that the areas with damaged mitochondria that is with low TMRE signal still harbor robust calcium sparks.
So the most difficult part of this procedure is to be able to obtain muscle fibers that are healthy of good quality, and also you need to be able to have a perfusion system allowing you to change the solution in a efficient manner. After watching this video, you should have a good understanding on how to use isolated muscle fiber to measure the calcium spark, which are the elemental units for calcium releasing skeletal muscle. And these calcium release events can certainly be used as a biomarker to evaluate the healthy status of muscle and the many pathophysiologic conditions such as muscle aging, diabetes, or muscular dystrophy.
